I’m trying to image the expression and localization of a fluorophore-tagged endogenous protein in the germline and it turns out that I need to isolate a gonad arm completely from the other tissues in order to eliminate the excessive signal associated with somatic expression. I have tried the protocol available on WormBook for gonad immuno-staining involving the use of two sharp needles in a scissor-like motion to cut the head of a paralysed worm. I’m able to successfully behead worms using one or both needles but rarely does it lead to extrusion of a gonad arm. Does anybody have any advice about how to do this better so that a gonad can be released and also separated from the remaining corpse prior to imaging? Any suggestions/tips are very welcome.
Thank you in advance!
have you tried using a hypotonic solution? like dissecting in 0.7X M9?
personally i find that a single 31.5 gauge needle works best for me.
We routinely dissect animals in 0.2mM levamisole (1xPBS + 0.0005% Triton) in a glass mortar to expose the intestine and gonad.
Hope this will help.
I found that cutting across the middle (at the vulva) worked better, possibly b/c you have a shot at two gonad arms per worm. I use one insulin syringe (27 or 28 gauge; smaller was too flexible for me) as the scalpel, a minimal drop (less floating) of PBS or sperm buffer (isotonic) without levamisole (residual thrashing may help to pop out the gonad, although I’ve never tested rigorously). And rely on the power of numbers - I dissect 50+ worms to get a few good ones for imaging.
Thank you all for your suggestions.
So far I was using an isotonic buffer (egg buffer) and 23 gauge needles (possibly, too big) or razor blades. I will try the various suggestions I received and see if I get an improvement! Thanks!
I think people use two hypodermic needles crossed like scissors, in a glass mortar as was mentioned. Play around with where in the body to dice the worm: I’d guess right behind the pharynx. But a lot of the germline labs routinely dice out gonad arms, so you should just approach them for the best technique.
The levamisole is important because it causes the body wall muscles to contract, helping to squeeze out the gonads after dissection with a needle.
We usually cut worms open in M9 on a polylysine treated slide for gonad staining. Keep practicing cutting their heads off right behind the pharynx, as this is most successful for us. Also, as stated above, cutting open 50+ worms usually gives me about 5-10 perfect gonads for imaging. Good luck!
Anne Campbell and Dustin Updike published a simple but effective method to separate worm gonads from carcasses with a capillary tube/mouth pipet. Works like a charm once you pull a good capillary - takes a little practice over a bunsen burner. Mouth pipetting may seem old-school but don’t knock it til you’ve tried it!
See Figure S1 in http://dev.biologists.org/content/142/10/1745.
Also, M9 is not a good/physiological buffer for worms or gonads - egg buffer, PBS, or even water is better. A little Tween-20 or other nonionic detergent (0.1% or less) helps to minimize gonad sticking.