TUNEL IHC help - technique help from experienced hands (Apr/20/2008 )
I am new to IHC and am doing TUNEL staining to look for apoptosis. I use a commercial kit from calbiochem and it is very straightforward. My problem is not in the protocol or method or principal but in carrying out the ACTUAL procedure. I fix my cells in 1% paraformaldehyde as per instructed but feel that I lose A LOT of cells because they are so hard to find. I look before I fix and they are visible but by the time I get to looking for fluorescence It is very difficult to see anything. Also, how can I differentiate between background staining and real staining? ANY advice on ANY aspect of this procedure would be so greatly appreciated as I am getting very frustrated with the results. A great thank you for anyone who can help. Thankssssss.
how do you attach the cells to the slide?
apes coated
baking overnight (post fix?)
or are you using coverslips and growing them on
apes coated
baking overnight (post fix?)
or are you using coverslips and growing them on
The cells are adherent so they attach to the chamber slide. So I fix with 1% paraformaldehyde for ten min at room temp wash with pbs 2x 5 min and post fix with ethanol:acetic acid solution for five min. Thats just what the protocol says to do. Is this correct? Thanks in advance.
why post fix - the pfa will quite happily kill em + freeze em in time - further dehydration then rehydration could compromised the cell attachment
also the shearing effect you get from tipping off a wash can damage tissue/cells - why not try single 7 min washes (except between primary and secondary - then do what makes you feel safest - astringent wash remember - admitidly i do single five min for all but i know my antibodies)
out of protocol - but as a wild card you could fix then put in 37'c oven over night (or hotter for shorter) then pbs and onto staining
dom
also the shearing effect you get from tipping off a wash can damage tissue/cells - why not try single 7 min washes (except between primary and secondary - then do what makes you feel safest - astringent wash remember - admitidly i do single five min for all but i know my antibodies)
out of protocol - but as a wild card you could fix then put in 37'c oven over night (or hotter for shorter) then pbs and onto staining
dom
Thanks for the advice! I am def. going to try that. I think minimizing washes within reason as you said is a great idea. Question, putting in the oven overnight just helps the cells adhere more strongly? Thanks again
i do it for fixed paraffin sections (again on apes) and used to flame blood smears to help em stick- be carefull if your antibodies are looking for heat sensitive epitopes (hence 37'oven - aka microbiology incubator).
like i said tho it is a wild card.
also make sure your pbs has tween (0.05%) as this helps it off the slide
dom
Thanks so much for your help dominic! I tried the assay with the few adjustments you said and it came out the best yet! I also put my cells in 37 degree incubator for two hours and think that helped a lot with the fixing. I was wondering though what is the purpose of post fixation? I did what you said and skipped it but am curious as to why people would even bother with this step? Any info would be much appreciated and any other tid bits of expertise would be great! Thanks again!
some people (not us obviously) read an experiment in a paper then copy it precicely not knowing why each step is in place - they then write a paper which other people copy etc etc. all it takes is for a few tweaks over time to be left in cos people dont know to remove them and hey presto - post fixing
after you fix tissue/cells it is refered to as fixed - why fix something thats already fixed (not broken? 
)
or i could be wrong and it could be vital to your experiment 
btw the heating doesnt help fixing - it helps attachment
glad to hear its going better
dom