Nasal Mucosa tissue coming off slides - (Apr/16/2008 )
I have started conducting IHC on nasal mucosa that were collected from hamsters perfused with McLean's Fixative (4% paraformaldehyde, 0.2M lysine buffer, 0.01% sodium periodate) and postfixed for 5-7 hours in this solution. Skulls were then immersed in 10% EDTA solution until they began to lose weight (usually 10-12 days). They were then cut coronally into four sections of approximately 2 mm each and then processed/embedded in paraffin. Afterwards, they are cut into 5 um thick sections and mounted on charged slides and placed in an incubator at 37 degrees celsius for 24 hours. At this point, tissues are deemed ready for IHC by deparaffinizing and rehydrating as usual through gradient alcohols and water. Since our lab studies prion diseases, we have optimized our protocol for detection of the pathogenic isoform of the prion protein (PrPsc) by denaturing the sections, using 10% formic acid followed by 6M guanidine. This is thought to denature the normal prion protein, leaving behind the resistant, pathogenic form, which we can detect using standard methods (primary Ab, Biotinylated secondary, Streptavidin-HRP, DAB, etc.). THE TROUBLE IS, the sections are coming off of the slides, sometimes before the denaturing protocols but not always. I tried baking the slides for 1 hour at 60 degrees celsius, with limited success...I am also trying baking for an additional hour to see if I can get those sections to stick better, but it doesn't look like there is much improvement...maybe a little...I will have the results tomorrow on how this may affect the IHC. ANYBODY HAVE ANY OTHER SUGGESTIONS ON HOW TO KEEP THESE TISSUES ON THEIR SLIDES? It would be much appreciated.
coat the slides with APES
bake in 37'C oven overnight
Thanks for the input Dominic. Several questions arise. Will this work if the tissue is already on the slide? I have several hundred of them cut already, so, it has to be something I can do post sectioning. Secondly, does this alter antigenicity? And, finally, what percent solution do you recommend? Thanks again.
clean slides in acetone (2min)
5ml apes in 250ml acetone (2min)
the baking can be done post - slow + low might be better that high + fast (also less damage to certain heat sensitive epitopes)
sometimes you just have to start again - try the apes on a few and it'll give you a hint as to if it can be fixed