This method for dissection and immunofluorescence is used by many C.elegans labs. It can be adopted to detect any protein that an antibody or tagged fusion protein is available for. Although it requires some practice to get this dissection right, this protocol is flexible and straightforward to troubleshoot.
For us, DAPI staining always works and we can usually find a condition that works for each antibody. The biggest challenge for this technique is the dissection step. You have to work quickly but confidently to get good germline extrusions.
Practice is key. To begin, place a holding slide on the stage of a dissecting microscope and place the coverslip on top of the holding slide. Then, pipette four microliters of the dissecting solution into the center of the coverslip and move the holding slide to one side of the stage to free up the view.
Pick 10 to 20 hermaphrodites from an NGM plate into the drop of dissecting solution. It's best to pick approximately 10 per slide but not so many that they cannot be dissected within five minutes. To dissect the animals, cross the points of the needles to make an X shape and use the bottom of the X to pin an adult down to the surface of the coverslip.
Next, position the X shape immediately behind the pharynx, about one fifth of the body length of a young adult or just below the clear part of the head. Then, decapitate the animal with one scissoring motion, releasing fully from the body cavity, along with one or both halves of the gut. To fix the sample with formaldehyde, pipette four microliters of the fix solution on the coverslip, very close to the drop with animals, while avoiding to pipette directly into the dissection drop and displacing the dissected carcasses.
Pin the coverslip to the holding slide using one finger. With the other hand, gently flick the coverslip a few times to mix the drops. Holding a positively-charged slide front side down, use it to pick up the coverslip in the center of the slide by gently touching the sample drop, and surface tension of the drop will pick up the coverslip.
Set the slide on the bench and fix for exactly five minutes at room temperature. During this time, label the slide with a pencil. To freeze crack the sample using liquid nitrogen while holding the labeled edge of the slide with tweezers, gently lower it into liquid nitrogen.
Release the slide, such that it leans against the side of the beaker with coverslip side up, and the slides are frozen within 10 seconds. If using dry ice to freeze, after scraping the frost off the surface of the aluminum block with a razor, firmly hold the labeled edge of the slide and set it on the cleared surface, applying some pressure downward to ensure full contact with the block. Freezing occurs within 10 seconds as the sample under the coverslip turns to ice.
Remove the frozen slide by firmly gripping the slide's labeled short edge, and brace one long edge against the bench. With the other hand, firmly grip a razor and slide the razor's edge down the slide to flick the coverslip off and away from the sample. Immediately place the slide in a Coplin jar containing 95%ethanol, and leave the slides in ethanol for at least five minutes.
Then, wash the slides in PBST three times for five minutes each at room temperature, while using fresh PBST for each wash. Fluorescence imaging results of germline nuclei demonstrated that DAPI binds strongly to DNA. Immunofluorescence staining was effective when the antibody targeting RAD-51, a marker for double-strand breaks, was present as discrete foci within miotic nuclei, co-localized on chromosomes marked by DAPI.
The kle-2 heterozygotes mutants have minor defects in chromosome structure, as shown by slightly disordered DAPI staining and an increase in double-strand break number, reflected in the higher number of RAD-51 foci. Both the dissection and freeze crack steps can be tricky so it's important to work quickly and smoothly. We recommend practicing these steps before attempting the entire protocol.
For example, you could practice dissecting in a larger volume of liquid, like 200 microliters, and gradually working down to our recommended volume of four microliters in a cover slip. This technique can be used for high-resolution imaging on a confocal or structured illumination microscope, or it can be used in combination with DNA or RNA-FISH, fluorescence in situ hybridization, to visualize how proteins localize in relation to specific sequences.
