- Methods for studying the DNA damage response in the Caenorhabdatis elegans germ line
- Research Overview
- N6-methyldeoxyadenine is a transgenerational epigenetic signal for mitochondrial stress adaptation
- Need advice?
- Molecules and Cells
Overall procedures of oxidative stress assays are very similar to those of lifespan assays, except for treatment with ROS-generating chemicals and time frame of the assays.
Methods for studying the DNA damage response in the Caenorhabdatis elegans germ line
Due to chemical toxicity, oxidative stress resistance assays are completed in relatively shorter times than lifespan assays. Thus, dead worms should be counted in short intervals e. Understanding the nature of specific ROS-generating chemicals is important for properly designing oxidative stress assays Keith et al. Paraquat, an organic compound used as an herbicide, produces superoxide anions and is widely used as an oxidative stress inducer. Hydrogen peroxide H 2 O 2 is a commonly used bleaching and decontaminating reagent. Tert-butyl hydroperoxide t-BOOH is a highly reactive and toxic organic peroxide that acts as a radical polymerization initiator.
Arsenite blocks pyruvate dehydrogenase and destroys energy production systems via increasing intracellular ROS levels. Juglone is a highly poisonous organic compound that forms a semiquinone radical and induces cell death by generating a large amount of superoxide anion radicals.
Surprisingly, recent studies have shown that low concentrations of oxidative stress-generating reagents increase lifespan in many organisms, including C. For example, we showed that low doses of paraquat 0. These results indicate an inverted U-shaped dose-response curve for paraquat and worm lifespan Hwang et al. Thus, choosing appropriate concentrations is essential for testing the effects of ROS-inducing reagents on lifespan and oxidative stress resistance. As oxygen is essential for aerobic organisms, animals are equipped with systems to adapt to different oxygen levels.
Hypoxia and hyperoxia refer to conditions in which biosystems are exposed to abnormally low and high levels of oxygen, respectively Rodriguez et al. This has led to the characterization of key genetic factors, including hypoxia-inducible factor 1 HIF-1 , the master regulator of cellular hypoxic responses Powell-Coffman, Hypoxia can be induced in two distinct ways, physical and chemical induction methods Jiang et al. For chemical induction, worms are treated with fresh 0. Worms exposed to hypoxia for a specific time are then transferred to NGM agar plates for recovery in room air, and numbers of dead worms are scored at regular intervals e.
Dead worms are then counted at regular intervals e. High environmental temperatures cause structural and functional impairments in macromolecules, wreaking havoc on animal physiology. Heat shock transcription factor-1 HSF-1 and the forkhead box O FOXO transcription factor DAF, which upregulate chaperone expression, reduce the accumulation of abnormal proteins and contribute to cellular protein homeostasis in C.
Overall, heat stress resistance assays provide foundations for identifying factors that regulate protein homeostasis. At this high temperature, C. Thermotolerance assays require very precise temperature control systems, as uneven temperature distribution in an incubator or a small temperature change between experiments may cause large variations in survival rates. For example, C. Cold tolerance assays using C. Dead worms are then counted similarly as described for other survival assays.
In natural environments, animals are exposed to various kinds of osmotic stresses. Hyperosmotic shock induces water efflux and protein aggregation, leading to body deterioration and protein homeostasis proteostasis Choe and Strange, ; Rodriguez et al. Organisms are equipped with several protection mechanisms that help maintain cellular osmotic homeostasis. These include increased levels of osmoregulatory solutes that function as chemical chaperones, including glycerol, sorbitol, inositol, and trehalose, and the induction of osmoprotective genes. Osmotic stress resistance assays using C.
The assays are executed to detect both acute 3 to 15 min and chronic responses hours to days to hyperosmotic stresses. Time intervals for worm counting should be determined depending on the concentrations of NaCl and characteristics of worm strains used. Ultraviolet radiation to nm is a major DNA-damaging environmental stress for most terrestrial organisms. UV stress induces DNA lesions and produces free radicals that damage other cellular macromolecules. Because UV radiation can cause harmful effects in humans, researchers should be cautious during UV light stress assays.
Liquid media culture systems are not suitable, as liquid absorbs UV and decreases its effective dosage. Ultraviolet light has dose-dependent effects on the health and survival of C. Survival against UV radiation can be performed using different stages of worms to determine its stage-specific effects. Protein homeostasis proteostasis is essential for cellular function and survival. Unfolded protein responses UPRs are elicited by many environmental stresses or genetic perturbations in the cytosol, ER, and mitochondria, and are key defense mechanisms for maintaining proteostasis.
Each UPR transmits signals from a specific cellular compartment to the nucleus. Evolutionarily conserved signaling factors, including IIS components, target of rapamycin, and AMP kinase, regulate proteostatic stress responses Vilchez et al. Accumulation of unfolded proteins in the ER acts as stress signals that are transmitted to the nucleus Walter and Ron, Therefore, tunicamycin results in accumulation of unfolded glycoproteins, which leads to ER stress. Typical C.
Tunicamycin is water insoluble and therefore should be dissolved by using dipolar solvents, such as dimethyl sulfoxide. Dithiothreitol is a reducing agent that blocks disulfide bond formation Cleland, and induces an immediate ER stress. Bioaccumulation of heavy metals causes severe diseases in humans, such as poisoning, renal dysfunction, and damage to the central nervous system Jaishankar et al.
Molecules that detoxify heavy metals include metallothionein proteins, which contain many cysteine residues and protect cells by chelating heavy metal ions Freedman et al. As these factors are evolutionarily well conserved, heavy metal resistance assays using C. For C. Because different mutants exhibit different lethal dosages, conditions should be optimized for specific experiments Barsyte et al. In addition, extensive care is needed when performing these assays, as heavy metals adversely affect human health.
Survival assays for pathogen resistance provide important information regarding host defense mechanisms and antagonistic relationships between pathogens and hosts. Understanding pathogenesis is also the basis for identifying prophylaxis and treatment strategies against infection. Various pathogens, including bacteria, have been used for pathogen resistance assays using C. Pathogenic bacteria are categorized into two groups, Gram-negative and -positive bacteria Darby, Pseudomonas aeruginosa , a ubiquitous Gram-negative bacterium that causes opportunistic infections, is the most popular model pathogen in C.
Salmonella species such as S. Serratia marcescens , Yersinia pestis , Photorhabdus luminescens , and Burkholderia pseudomallei are other examples of Gram-negative bacterial pathogens of interest. Gram-positive pathogenic bacteria, such as Staphylococcus aureus , Enterococcus faecalis, Enterococcus faecium , Streptococcus pneumoniae , and Microbacterium nematophilum are used for pathogen resistance assays. Because the general methods for most C. First, bacterial colonization in the intestine kills C. Second, P. These two mechanisms have been used for establishing slow- and fast-killing assays.
For slow-killing assays, PA14, the most commonly used P. Another recently developed assay relies on a nutrient-poor liquid culture killing assay in which worms are killed within 48 h by PA14 in liquid medium Kirienko et al. Among the assays described above, slow-killing assays are the most popular survival experiments using P. Plates for small-lawn assays, which are more popular than big-lawn assays, are prepared by dropping small volumes of bacteria on plates e. Plates for big-lawn assays are prepared by spreading large volumes of PA14 e.
Unlike small-lawn assays, no PAfree space is present on big-lawn assay plates where worms can avoid PA Performing both small- and big-lawn PA14 resistance assays can help identify different mechanisms of pathogen resistance i. Several specific considerations for slow-killing PA14 survival assays should be noted. First, concentrations of PA14 affect virulence of the bacteria due to quorum sensing Papenfort and Bassler, Therefore, proper cultivation temperatures and media composition, as well as the preparation of fresh bacterial lawns, are crucial for performing reliable assays.
Second, using worms in proper developmental stages is critical; young adults and L4 larvae are routinely used for the assays Darby, ; Keith et al. Third, due to prevalent internal hatching of the worms, FUdR treatment is highly recommended. Fourth, dead worms should be counted every 6 to 12 hours because they quickly lyse and become transparent due to exoenzymes produced by PA Opportunistic fungal pathogens, which are capable of infecting and killing C.
These include Cryptococcus neoformans , Candida albicans , and Drechmeria coniospora. Studies regarding fungal pathogenesis by using C.
- The Potty Partner - Riddles, Tests and Lists to Live By.
- Common Ground.
- Table of Contents.
- Alkoholabhängigkeit bei Jugendlichen (German Edition).
- A Collection of Poems.
- Stimulus: American Recovery and Reinvestment Act of 2009: PL 111-5: Official Text (Stimulus American Recovery and Reinvestment Act of 2009);
- Fix Your Marriage: 10 Simple Steps To Put The Joy And Intimacy Back In Your Marriage (One New Habit).
Here, we describe worm survival assays using C. Various virulence factors, such as laccase and a polysaccharide capsule, and signal transduction cascades that affect virulence have been identified. The fungi kill C. For pathogen resistance assays, C. The microsporidian Nematocida parisii is an intracellular pathogen that kills C. The N. Interestingly, the responses of C. These findings have led to interesting perspectives regarding comparative pathogenicity and immunity.
For N. Similar to other pathogen resistance assays, L4 larvae or young adults are used for the survival assays at appropriate intervals e. Upon completion of survival assays, data should be analyzed with proper statistical methods. Two widely used curves for survival analysis are simple survival curves and mortality rate curves. The most commonly used survival curves are Kaplan-Meier survival plots, which illustrate the percent of live animals against time Kaplan and Meier, The log-rank test is used for comparing the survival distributions under two conditions and yields average survival times and P- values.
Mortality rate curves are drawn by calculating mortality rates, which are obtained by dividing changes in death incidence by time, and are used for deducing causes of deaths: accumulation of irreversible damage vs. Several bioinformatic tools used for drawing these curves and for statistical analysis have been developed; these include open-source programs, such as online application for survival analysis OASIS Yang et al.
In this review, we described survival assays using populations of C. The molecular and cellular basis for the regulatory mechanisms of C. Likewise, pathogen resistance assays have enabled understanding of pathogenecity and immunity through the identification of critical virulence factors and immune—regulatory factors Ewbank and Pujol, ; Kim and Ewbank, In addition, studies indicate that survival assays using isogenic populations of C. As shown by numerous important discoveries made in the last 40 years, survival assays using C. Title Author Keyword Volume Vol.
Hae-Eun H. Received February 6, ; Accepted February 23, All rights reserved. Keywords : aging, C. Solid Culture Systems Agar-based solid culture systems are the most common C. Liquid Culture Systems Media for liquid culture-based C. Common Features Between Solid and Liquid Lifespan Assay Systems For both solid- and liquid-based lifespan assays, separating adult hermaphroditic worms from their progeny is a significant challenge, as progeny that have reached adulthood are difficult to distinguish from their mothers.
Automated Lifespan Sssays The conventional lifespan assays described above are manual counting assays that have several limitations; for example, manual assays are labor intensive and prone to researcher-oriented bias, and worms are vulnerable to mechanical and heat stresses induced by platinum wire usage. Oxidative Stress Survival assays under oxidative stress conditions provide information regarding biological systems crucial for responding to oxidative stresses. Hypoxic and Hyperoxic Stresses As oxygen is essential for aerobic organisms, animals are equipped with systems to adapt to different oxygen levels.
Heat Stress High environmental temperatures cause structural and functional impairments in macromolecules, wreaking havoc on animal physiology. Cold Tolerance C. Osmotic Stress In natural environments, animals are exposed to various kinds of osmotic stresses. UV Stress Ultraviolet radiation to nm is a major DNA-damaging environmental stress for most terrestrial organisms.
ER Unfolded Protein Stress Protein homeostasis proteostasis is essential for cellular function and survival. Bacterial Pathogens Pathogenic bacteria are categorized into two groups, Gram-negative and -positive bacteria Darby, Fungi Opportunistic fungal pathogens, which are capable of infecting and killing C.
Intracellular Parasites The microsporidian Nematocida parisii is an intracellular pathogen that kills C. Lifespan assays are either performed with solid or liquid media. Solid culture systems using agar-based media are a major method for lifespan assays. Liquid culture systems using S-media are widely used for testing the effects of chemicals on lifespan. Synchronized worms at a specific developmental stage are transferred for manual or automatic counting, and results are then analyzed using various statistical tools. Shown are various stress-inducing agents with typical assay conditions.
Shown are representative pathogenic bacteria, fungi, and parasites used for C. Some organisms, such as C. Other organisms, such as humans, have variable lineages and somatic cell numbers. As one of the first pioneers of cell lineage, in the s Dr. Sydney Brenner first began observing cell differentiation and succession in the nematode Caenorhabditis elegans. Brenner chose this organism due to its transparent body, quick reproduction, ease of access, and small size which made it ideal for following cell lineage under a microscope. By , Dr. Brenner and his associate, Dr.
John Sulston , had identified part of the cell lineage in the developing nervous system of C. Recurring results showed that the nematode was eutelic each individual experiences the same differentiation pathways. This research led to the initial observations of programmed cell death, or apoptosis.
- College Adult Picture Babes Extremely Sexy And Stunning (Adult Picture Book Book 3);
- The Ends of Solidarity: Discourse Theory in Ethics and Politics (SUNY series in Contemporary Continental Philosophy).
- Laser microsurgery in Caenorhabditis elegans.!
- Laser microsurgery in Caenorhabditis elegans..
- Navigation menu.
After mapping various sections of the C. Brenner and his associates were able to piece together the first complete and reproducible fate map of cell lineage. They later received the Nobel prize for their work in genetic regulation of organ development and programmed cell death. Examining protein localization using GFP or DsRed fusion proteins requires injection of corresponding transgenes into animals.
Thus, a number of caveats exist in interpreting results from such experiments. For example, expression from transgene arrays is often higher than from the endogenous locus. Furthermore, fluorescent proteins are fairly bulky, thus, fusion proteins may not display appropriate localization. In addition, promoter elements used to drive expression of GFP transgenes are often expressed in cells where the endogenous protein is normally not made, or vice versa. Fluorescently-labelled antibodies generated against a protein of choice allow direct examination of endogenous unmodified proteins, giving a clearer assessment of where endogenous expression is localized.
Antibodies coupled to gold particles can also be used for electron microscopy to examine subcellular localization in great detail. Antibodies can only label fixed tissues, thus, they cannot be used for real time observation of protein localization dynamics. In addition, fixation often destroys cellular structure, limiting the resolution obtained by immunostaining. Detailed protocols for use of antibodies can be found in the gene expression chapter. A number of microscopes are now commercially available for generating and examining fluorescent signals. All are able to resolve fluorescent entities both in space and in time.
Differences among these microscopes center on spatial and temporal resolving power. This binocular microscope allows visualization of highly expressed fluorescent signals at low magnification. The major use of the instrument is to visualize GFP and DsRed-labelled proteins in living worms on the surface of standard agar plates.
Generally, use of this microscope allows selection of animals expressing a fusion protein of interest, as well as examination of very coarse cellular features e. This method does not allow fine structure to be examined. The microscope consists of a standard binocular dissecting microscope equipped with epifluorescence. This microscope has significantly higher resolution and magnification compared to the dissecting fluorescence microscope, and observation can be combined with DIC Figure 5.
Objectives of X are often used, and allow examination of gross subcellular localization of proteins. Images collected from such a microscope at high magnification suffer from an extensive background of fluorescent light emitted by cells not in the plane of focus and of scattered light.
This often severely cuts down on the resolving power of this microscope. Figure 5. Standard fluorescence microscope image of the amphid ASE neuron and sheath cell top. This instrument is essentially a compound fluorescence microscope equipped with a sensitive light detection system. Light collected from optical sections through the sample is processed using sophisticated computer software, which assigns out-of-focus light to its correct focal plane Wallace et al. Images generated by this microscope consist of optical sections through a sample, and allow 3-dimensional reconstruction of the fluorescent signal with little background.
Sections can also be projected onto a single image, giving highly resolved artificial two-dimensional images Figure 6. All images are viewed on a computer screen. Time-resolution of the technique is, in general, inversely proportional to spatial resolution. Thus, the more optical sections imaged, the larger the time intervals between image collections at any given focal plane. This type of microscope is not efficient for examining processes that occur over intervals of seconds or faster.
Figure 6. This instrument consists of a compound microscope equipped with a laser for fluorophore excitation, and a special detection set up consisting of a small pinhole through which emitted fluorescence light produced only near the focal plane of observation can pass. Light passing through the pinhole is collected in a sensitive light detector, and images are produced on a computer screen. As with the deconvolution microscope, images consist of optical sections through the sample.
Time-resolution of this microscopy is affected by several factors, including the time required for the laser to scan the field of view, and the amount of light that needs to be collected to visualize fluorescence. Unlike the deconvolution microscope, which uses all the light emitted by the sample to calculate the point of emission, a confocal microscope only collects light traveling through the pinhole, necessitating longer exposures or more sensitive light-detection systems.
This instrument is a variation of the laser scanning confocal microscope.
Here, many pinholes are arranged in a spiral pattern on a disc that can rotate at high speed. Thus, time resolution can be excellent on the order of l00 msec or less , and photobleaching is generally not a significant issue as with other microscopes Nakano, Image display is essentially as with the standard confocal microscope Figure 7.
Figure 7. The output from this microscope is similar to that from deconvolution or confocal microscopes. Thus, optical slices of images are generated that can be reconstructed to form a three dimensional image. Multiphoton microscopy eliminates out-of-focus light by directing excitation to the focal plane. This is accomplished by shining photons of long wavelengths and at high density onto the sample.
At the focal plane long wavelength photons can superimpose to become, effectively, a shorter wavelength photon. This photon can now excite the fluorophore and induce fluorescence. Superposition of photons occurs significantly only at the focal plane. Multiphoton microscopy is also useful for looking at samples that are much thicker than confocal or deconvolution samples, with nearly equivalent resolution Helmchen and Denk, ; Michalet et al. Electron microscopy EM is currently the method of choice for examining subcellular structures in C. The resolution offered by the technique is unparalleled, allowing objects as small as, or even smaller than ribosomes to be viewed.
Although EM is technically demanding, it is well worth the effort if resolution of small structures is of importance. Transmission EM TEM generally involves fixation of animals with any combination of gluteraldehyde, paraformaldehyde, or osmium tetraoxide OsO 4 , embedding of fixed animals in a special resin, sectioning animals sections are usually 50— nm thick , staining, and imaging on an electron microscope Hall, ; Figures 8 and 9. Fixation conditions are generally the key to a successful EM experiment.
Standard treatments with fixatives are adequate for most applications, however, they have the disadvantage that cellular structures are often slightly distorted because of osmotic imbalance and other damage that occurs during the fixation procedure. Tissues and cells are generally rounder, and the ultrastructure seems to reflect the in vivo structures more accurately Figure Because HPF fixation has only recently come to prominence in C.
Specifically, results are often more variable than using standard fixation. In addition, embryos and L1 larvae seem to fix better than older stages. Interestingly, embryo fixation using standard methods is fairly difficult, thus, HPF and standard techniques are somewhat complementary. Figure 8. TEM micrograph of a longitudonal section of the C. Figure 9. TEM micrograph of cross-section of C. Figure Because fixation is so critical for good ultrastructure, we present below several alternative protocols for TEM.
In this method, whole-mount animals or structures are viewed by EM, offering both broad-scale and highly resolved images Figure SEM of a dissected C. The protocols below are intended to give a sense of the types of fixation procedures used for EM and should be used as guidlines for those with previous EM experience.
These protocols are not intended for teaching EM from scratch. Fix in 2. Cut out small agarose blocks containing worms; should be large enough to be excluded from pipette tip, but small enough to fit into embedding mold. Dehydrate through ethanol series: 5 min. Place samples in fresh resin at room temp; place samples in air-tight containers and agitate or rotate samples slowly. Rotation or agitation of the samples helps with the infiltration.
Mix components well with wooden applicator, add accelerator, 1. Place resin into dessicator and put under vacuum for 5—15 min to remove gasses from mixture before using. Rinse worms off plates with M9, then substitute with fixative: 0. Let sit on ice for 1 hr in the dark, cut worms open close to tissue of interest, rinse 3x with 0.
Place 5—6 worms in a very small drop of buffer, withdraw the buffer and quickly place an agar drop over the worms and align worms with a hot worm-pick as closely as possible. Place gelatin blocks into flat transparent embedding molds, blocks can be aligned according to cutting requirements, place mutant no. Trim blocks and cut worms to the desired level with glass knives. Cut thin sections of light gray to white color. Long continuous ribbon of sections can then be picked up with a coated grid, where the sections according to the length of the slotted grid can be separated by eyelashes.
Hold the ribbon of sections with eyelash in the left hand and pick up sections by submerging the grid underneath the water surface in the trough and then slowly come up at an angle. Fix in 0. After 30 min. First change is in 1 part resin to 2 parts propylene oxide, allow to sit 3—4 hours or more. Second change is in 2 parts resin to 1 part propylene oxide, for 3—4 hours or more, 1 part propylene oxide to 2 parts resin overnight, rotating. The next day, place samples in fresh resin at room temperature; place samples in air-tight containers and agitate or rotate samples slowly during infiltration.
Then change into fresh resin at least 3—4 times, 2—4 hours per change. Resin formula [use pre-dessicated plastic beaker with gradations for easy measurements]:. Add fixative 2.
- Methods in cell biology - WormBook - NCBI Bookshelf!
- Haikus sin estación (Spanish Edition).
- Nektarios Tavernarakis.
- 2. Visualizing cells and their components.
- Developmental and Neuronal Cell Biology.
After microwave bouts, continue soaking animals in same fix at room temp for one hour. Change is in 2 parts resin to 1 part propylene oxide, for 3—4 hours or more, 1 part propylene oxide to 2 parts resin overnight, rotating. Scientific microwave oven we use a model from Ted Pella ; kitchen ovens do not properly control the microwave energy pulses for this purpose. See Paupard et al. Scoop animals off an NGM plate in a coating of E. Place the animals and bacteria into a metal hat until interior compartment is full to the brim.
N6-methyldeoxyadenine is a transgenerational epigenetic signal for mitochondrial stress adaptation
Place a blank hat on top to seal the compartment and load into HPF machine to freeze. There should be no air space surrounding the sample within the metal hat [to obtain maximum pressure]. The next day, place samples in fresh resin plus accelerator at room temperature and place infiltrated worms in eppendorf tubes or gelatin capsules. If desired, cut best worms out of embedment and re-embed in resin at desired orientation and re-cure in sealed flat mold.
High Pressure Freeze apparatus; a very expensive item, but they are available at several national facilities including those in Madison, Minneapolis, Berkeley, and Albany. Freeze substitution apparatus; can be home-built or purchased. As with any ultra-rapid freezing method, the principal concern is optimizing the specimen geometry to maximize the heat transfer rate with the coolant used. Increasing the heat transfer rate is often best achieved by minimizing the size of the specimen chamber in the direction that the coolant is being applied.
The Bal-Tec HPM high-pressure freezing device has specimen holders that can be used to create specimen chambers that closely match the diameter of the specific C. The most suitable spacers found so far are slot, hole and Chien EM grids ordered from Ted Pella, but probably available from other suppliers; thicknesses were measured with a Fowler micrometer. Multiple grids can be used in combination to create specimen chambers of other thicknesses as needed:. On the flat side of the Type B holder m planchette , place a grid suitable in thickness to the worms being frozen. Place a dab of E.
Briefly touching the grid to a thin E. That's plenty for sectioning in case the freezing is good, and not too many that if the freezing is bad that a lot effort has been wasted. Fill the rest of the well with E. The consistency of the packing material is important. You want to avoid packing material that is too soupy because the higher water content does affect the freezing rate.
Under LN2, the frozen sample is split open with the tips of a pair of forceps previously cooled in LN2. Transfer the holder s containing the sample to a 2. Transfer the vial to your freeze-substitution device. This typically gives good preservation and enhanced membrane contrast with well-frozen samples. There is, however, wide latitude in temperatures, warming rates, and durations of each step that give excellent results. In this case it is helpful to have a:. The insulated container should be allowed to cool down prior to loading samples for substitution, by filling it with dry ice about 30 minutes before use.
The cryovials are transferred to the aluminum holder in a styrofoam container, or dewar, previously equilibrated with LN2. Once all the sample vials are loaded, the aluminum holder is placed at a slight angle in the picnic container holding the dry ice, and some aluminum foil is wrapped around the block to keep the sample vials secured during substitution. The thermocouple is positioned through a small hole in the aluminum foil to a spot close to the sample vials, and the lid of the picnic cooler closed.
The picnic cooler is placed on the orbital shaker and fastened to the platform of the shaker with bungee cords.
The shaker is turned on and set at 50— rpm. The substitution with this agitation is performed for 3—4 days, with replenishment of dry ice as needed to keep the specimen covered with dry ice. Fill the cooler for the last time with dry ice 48 hours before the planned end of the substitution time. Over the 48 hours the dry ice will gradually disappear and the aluminum block will gradually warm. Remove the specimens at the desired end point temperature of the substitution and rinse the specimens with acetone at that temperature. Infiltration should be performed using a tissue rotator whenever possible.
At the completion of the freeze-substitution program, rinse the samples 2x with dry acetone, and then exchange the acetone for propylene oxide. Infiltrate the samples with propylene oxide:resin for 30 minutes followed by resin:propylene oxide for 30 minutes, and then transfer to pure resin containing catalyst. An additional 2—3 changes of resin, 1—2 hours each, followed by an overnight change in resin will ensure complete infiltration of the worm. All infiltration steps should be performed with a tissue rotator. It is fine to leave the worms in the packing material, but it is a little harder to see the them to orient for sectioning.
In order to have maximum control over the orientation of the worms for sectioning, I prepare blocks for sectioning in 2 stages. First, I flat embed the worms between 2 glass slides previously coated with spray-on Teflon Cat. Previously coat one side of each pair of glass slides, allow to dry, and then wipe off the white residue with a Kimwipe. A doubled-over piece of Parafilm at each end of the slide works well as a spacer to prevent the worms from being crushed.
After polymerization, a razor blade is used to gently pry the slides apart. Using a razor blade, cut out the worms to be sectioned and re-embed them in regular molds in the desired orientation. The teflon does not seem to interfere with the adhesion of the resin containing the worm with the fresh resin it is re-embedded in. I have not experienced separation at this junction during thin sectioning.
Rinse the samples 2x with dry acetone and then infiltrate with your immuno-EM resin of choice as usual. The trick is to add and dissolve 1. LR White polymerization is very sensitive to oxygen. To avoid getting an unpolymerized film of resin at the top of the mold, I place a strip of ACLAR film Ted Pella on top of the mold, taking care not to trap a bubble of air, after I put the worm in and fill the mold with resin.
Make up above mixture immediately before mounting embryos. The pad should be thick, so use triple thickness tape when preparing slide. Transfer the eggs to pads with minimal fluid to preserve osmolarity. Transfer with a pick or mouth pipette. Use an eyebrow hair to push the eggs to the center and arrange them so that they are close to each other without touching.
Overlay a small amount of slightly cooled agarose mix on slide and place a coverslip on top. Try either 12 mm or 18 mm coverslips. Make a map of the embryos- their arrangement on the pad, their orientation, and their relative stages. Mount a bunch of 1, 2, and 4 cell embryos on a pad, then watch them go through landmark events, like gastrulation, the migration of the dorsal hyp nuclei, or cell deaths to stage them. When an embryo reaches the stage you want, permeabilize the egg shell and vitelline membrane with a laser.
Shoot for edge of shell. You can see the break in the shell and the membrane. The laser needs to be at full power. Before fixing the final specimens, try several trial embryos with differing amounts of sucrose in the final fixative. While watching under the microscope, shoot several laser holes in the eggshell. If the embryo collapses rapidly implodes , then the sucrose concentration is too high.
If the embryo blows up rapidly explodes , then the sucrose is too low. Under optimal conditions, the embryo will be seen to swell in volume very slowly after a laser hole is put through the eggshell. Thus the final fixative should be tested empirically on the day of fixation, to optimize the exact osmolarity. Eggs will usually stay in place. Begin dehydrations and washes. Either leave the group in their original agar pads, or cut up the pad and place individual eggs in separate wells of a 9-well glass plate. Either way, trim the agarose with a clean new razor blade. Wash in 0.
The larger blocks help protect the embryos as you go through all the steps and they are also much easier to see and therefore to avoid sucking into your pipette and discarding. The distinct shapes allow you to dehydrate all the embryos in the same vial without confusing them. Transfer blocks to whatever mold you will use to hold the embryo and try your best to orient embryo for easiest trimming and cutting. Recover the gonads by centrifugation at rpm in a IEC clinical centrifuge for 2 min. After digestion place gonads on ice and gently remove supernatant from gonads which had settled during incubation period.
Because gonads are fragile until fixed, add all solutions slowly. Wash gonads with cold 1x PBS, allowing the gonads to settle by gravity. After they are fixed, wash the gonads three times with cold 0. After the final wash pipette the gonads into a watch glass and hand select intact gonads under a dissecting scope. Place gonads in the cooled chamber of a critical point drier with the samples being covered with ethanol. To prevent the gonads from being damaged when the solvent enters or is vented from the chamber, place the gonads between several layers of filter paper cut with a standard hole punch.
Transfer dried samples to aluminum stubs covered with double stick tape using a camel-hair brush. An important task in deciphering protein function is the identification of other entities with which it interacts. Below are protocols describing immunoprecipitation IP and chromatin immunoprecipitation ChIP that should serve as general guidelines for in vivo interaction studies in C. These interactions can often be confirmed by standard in vitro techniques such as two-hybrid, GST pull-down studies, and electrophoresis mobility shift assays EMSA. Float adults worms off 9-cm NGM plates with 5 ml of M9.
Approximately 6—8 plates saturated with asynchronous population of worms, but not starved, will be needed to seed each liter of liquid culture. Add 10—15 ml of saturated HB and monitor the food supply at least once per day. Spot 1 drop of culture onto a 5-cm NGM plate and allow the liquid to evaporate for about 1—2 minutes until the worms can crawl. If there is sufficient food, the worms should not scatter or forage. Alternatively, starved worms appear somewhat translucent. Treat each liter of culture with ml of freshly made alkaline-bleach solution.
Mix on a stir-plate for 5—10 minutes for mutant worms, it may be advisable to bleach for less than 5 minutes. Stop the bleaching process when the adult worms start to break open. Centrifuge the bleached worms at 1—2, rpm in a tabletop centrifuge. Stop the centrifuge as soon as the speed reaches 2, rpm.
It takes several minutes for the rotor to come to a complete stop. Resuspend the worm in M9 and centrifuge as in step 5. Repeat this wash step once more for a total of two M9 washes. Seed 0. Generally use 0. Allow the embryos to hatch overnight without food and feed the synchronized L1 larvae the next day. Wait 1 minute in between bursts for cooling. Collect the supernatant in a clean mL tube. Sonicate as described in step 2 to shear the DNA. Collect and quick-freeze the supernatant in 0. Thaw embryo lysates on ice.
Spin in a microcentrifuge for 2 min. Transfer the supernatant to a new microfuge tube and spin in a microcentrifuge at top speed for 10 min. Use the supernatant for IP. Bring the final volume up to 1. Pellet the antibody-antigen complexes captured on the Protein A Sepharose beads by spinning in a microcentrifuge for 2 min.
Remove the supernatant. Wash the beads by adding 1 mL of ChIP buffer and spinning in a microcentrifuge for 2 min. Remove the wash buffer. Repeat this step for a total of four washes. Pellet the beads as described above, remove and save the supernatant. Precipitate the eluate with trichloroacetic acids. Harvest embryos by bleaching gravid hermaphrodites. Wash the embryos once in the formaldehyde solution. Aspirate away the wash solution. Add fresh formaldehyde solution to 50 mL and gently shake using a nutator at room temp for 30 minutes. Wash the cross-linked embryos once with 50 mL of 0.
Wash the embryos once with homogenization buffer. Spin to pellet the beads. Transfer eluate to a clean microfuge tube. Repeat elution step once more. Phenol-chloroform extractions. EtOH precipitate. Autoclave to sterilize. Trace metals solution: Per liter solution, add 1.
Autoclave to sterilize and store in the dark. Add KCl to the desired concentration. This protocol is a modified version of a protocol from Upstate Biotechnologies www. There is no harm in scaling up for cleaner results, especially with poor antibodies. Adult N2 worms grown on either HB or RNAi expressing bacteria are bleached and washed before being immersed and rotated in 1. Embryos are washed extensively with M9 at least 3 times and gently centrifuged. Be careful not to rupture embryos. Do not over heat or allow to foam excessively.
Keep on ice. Centrifuge and wash the beads once at room temperature with constant rotation with each of the following buffers Note: You can make these buffers, see Upstate recipe, but I find that the beads are cleaner when these products are used : 1. After the last wash, the beads are washed three times with 1X TE, pH 8. Keep the supernatant and repeat this step once more.
The samples are now ready for PCR reactions. Endocytosis has been studied in two cell types in C. Coelomocyte endocytosis is usually assayed by uptake of proteins conjugated to dyes that are injected into the body cavity. Oocyte endocytosis is measured by uptake of GFP-tagged yolk protein secreted by the intestine.
Below are protocols for both assays. Insert a needle into the body cavity in the pharyngeal region. You should see liquid flowing in the body cavity. Recover worms onto seeded plates with Egg buffer and incubate for appropriate time. Incubate slides on ice for at least 20 min. Slides are kept on ice until just before starting observation. Usually, about 10 worms are injected for each time point.
Using transgenic worms expressing GFP fusions of endocytic markers in coelomocytes helps to find coelomocytes. Alternatively, wash worms off a seeded plate with 1. L1 progeny of these worms present during dissection do not interfere with anything. Suspend washed worms from one plate in 0. Depression slides work well. Use the deepest you can find. As paralysis sets in, begin cutting off heads at level of the pharynx. To cut off the head, place the head between two 25 gauge syringe needles and decapitate by moving needles in a scissors motion avoid needles with bent tips.
For most animals, at least one gonad arm and the intestine should extrude completely. Fix the cut worms by adding 1 ml of 1. Rock at RT for 10 min. GFP autofluorescence survives the aldehyde fix well, but probably not the methanol. Change buffer at least 2X. Cut worms are spun down for 30 sec to 1 min at 4, rpm between buffer changes. Spin down cut worms, remove supernatant, add primary Ab diluted in PTB. You can never wash too much. Spin down cut worms, remove supernatant, resuspend in a glycerol antifade reagent such as Slowfade Light from Molecular Probes.
Mount on pads of Permanent Springtime Agarose just before viewing. Can seal with nail polish. Mix 0. Mix until dissolved. Add dilute HCl until pH drops to between 6 and 8 as judged by pH paper. Add 5 ml 10X PBS. Bring up to 50ml final volume with dH2O. Use the same day. Fresh dilutions of concentrated EM grade formaldehyde from sealed ampules is also acceptable.
Specific chromosomal sequences, as well as chromosome-associated proteins can be elegantly labeled in C. In general, signals are easier to detect and resolve in larger nuclei, thus, best results are obtained by looking at germ cells, intestinal cells, and cells in the early embryo.
A variety of protocols related to chromatin visualization are presented below. Pachetene stage germ cells stained using FISH. Protocol Antibody staining of C. To release the embryos, cut at the vulva. Place the slide in a humid chamber for 5 minutes. Freeze the slide on a piece of dry ice. Let the slide sit on the dry ice for at least 10 minutes. Remove the slide from the dry ice and quickly pop off the cover glass with a quick downward stroke of a single-edged razor.
Wick off most of the PBST, but do not allow the worms to dry. Place the slide in a humid chamber. Incubate overnight at room temperature. Incubate the slide in a vessel containing PBST for at least 10 minutes. Carefully remove the piece of parafilm. Incubate the slide in a vessel containing fresh PBST for at least 10 minutes.
Molecules and Cells
Repeat this step once. Keep the slides in the dark whenever possible. Seal the slide with fingernail polish. Observe the slide. When trying an antibody for the first time, it is often good to try all three fixative concentrations for 5 minutes each Step 1. This gives you a baseline for adjusting the protocol for your specific antibody.
You might notice certain trends. For example, when you use lower fixative concentrations, you might find that the antibody staining becomes brighter, but the structure of the chromosomes becomes worse. As you use higher fixative concentrations, the antibody staining might be worse dimmer, penetration not as good , but the structure might be better.
If this is the case and you need the chromosomes to have better structure, you might try incubating in a lower fixative concentration for a longer time period or in a higher fixative concentration for less time. If penetration is not good with the lowest fixative concentration that you tried, you might try an even lower concentration, or you might try less time in the lower fixative concentration e. The fixative concentration, fixation time, and freeze-cracking step Step 2 are very important.
You can see great differences in staining with small changes in fixative concentration and fixation time, and it is important to get a good freeze-crack. You can tell that the penetration of the antibody was not good when nothing stains or just the outer surface or outer layer of cells stains. If you see that worms on one part of a slide stain but worms on another part do not stain, you may be having freeze-cracking problems Step 2. If you leave the slides on the dry ice for too long Step 1d , the slides can get icy, and you might not get a good freeze-crack Step 2.
For a new antibody, try a concentration gradient with the antibody. The staining is sometimes greatly improved with lower concentrations of some antibodies. Removing the ethanol step Step 3 can produce dramatic changes in the antibody staining. It is good to try the protocol with and without the ethanol step. Ethanol is a coagulating fixative that denatures proteins and changes the protoplasm into an artificially interconnected network. This protocol does not have any block. First, try a new antibody without block to see everything that the antibody sticks to without block.
You might find that block is not needed or that block causes greater background staining rather than lower background staining. Swirl the slide around a little after adding the fixative in Step 1c. You can place the cover glass on the slide shortly after mixing the fixative and the sperm salt solution together in Step 1d. This can help to get the worms to stick to the slides better. The incubation times with both the primary and the secondary antibodies can often be less.
For example, you can try incubating in secondary antibody for four hours at room temperature. Several different anti-fade solutions can be used, e.