Does anyone have a protocol for immunostaining whole worms with anti-Flag antibodies? I’d like to know what fixation, permeabilization and staining conditions work best for that particular antibody.
Thanks!
Dan
Hey Dan,
I am currently doing Anti-flag immunostaining, however I do them on embryos.
I am using the M2 anti-flag antibody from sigma Here.
It works pretty well, i got some pretty good signal with the antibody diluted 1/1000 but I think that you will have to find the best dilution for your staining, mine is a bit particular.
My fixation protocol is close to the one I use for smFISH which is:
Fixation with 4% PAF in PBS for 15 minutes followed by a snap freeze in liq N2 for 1 minute
Thaw the samples in water and place them for 20 minutes on ice.
Rince samples 3 time with PBS.
Resuspend samples in 70% EtOH - place them at 4°C O/N (samples can be kept in fridge for several days)
Rince samples with PBS (2-3 times)
Permeabilization with PBS containing 0.5% Triton X-100 for 5 minutes @ RT (I also tried Methanol at -20°C I did no see major differences compare to Triton)
Wash samples twice with PBST.
Blocking
Add 4% BSA in PBST (=Blocking solution) to samples and incubate for 10 min at room temperature
Then add antibody diluted in blocking solution - incubate overnight 4°C
As secondary antibody I use a monoclonal anti-mouse coupled to Alexa 488 diluted max 1/2000
I hope this help, if you need more details do not hesitate to ask
Hi Thomas,
Thanks for your reply. Just a couple of follow-up questions:
-
What’s the purpose of the 15 minute incubation prior to freezing? I’d have guessed that the PFA wouldn’t get past the eggshell until after the freezing, but maybe I’m wrong about that.
-
Are you doing this protocol on slides or in tubes? If in tubes, at what stage to you transfer the embryos to slides?
Thanks again for your help. If anybody else is reading, I’d still be very interested to hear from anyone who’s done this with whole worms.
Dan
To be honest I don’t really know what is the purpose of these 15 minutes incubation. As I told you I am following the fixation protocol provided by stellaris to make smFISH experiments HERE, maybe this step can be removed from the protocol and your signal will be better.
All the steps are done in tube. In the end after the last wash, I remove as much liquid I can to concentrate the embryos.
Then I mount them on slides with an anti-fading reagent and I seal the coverslip with nail polish.
Using this protocol I could stain nuclear membrane proteins, histone, and transcription factors.
This protocol is pretty simple, but it is not impossible that doing staining directly on slides could give better results.
The only certainty I have is that the FLAG antibody I suggested works pretty well.
In my grad lab, we used the M2 antibody as well following a modified Finney-Ruvkun fixation (it’s essentially formaldehyde-methanol fix). PM me your email and I’ll send you the protocol (I’ll have to scan it and send as a PDF).
Steve
- What's the purpose of the 15 minute incubation prior to freezing? I'd have guessed that the PFA wouldn't get past the eggshell until after the freezing, but maybe I'm wrong about that.
To be honest I don't really know what is the purpose of these 15 minutes incubation. As I told you I am following the fixation protocol provided by stellaris to make smFISH experiments HERE, maybe this step can be removed from the protocol and your signal will be better.
I believe the answer relates to the original protocol for smFISH technique developed in Sanjay Tyagi’s lab and developed further by Arjun Raj. They added a short fixation step prior to freeze-cracking embryos to increase the effectiveness of the latter (by making them more brittle I assume).
Steve