I have cryo-sections on superfrost slides that were never fixed (fresh frozen) and I want to fixate it in order to use histochemistry method that only works on fixated tissues (black-gold stain for myelin). Is there any way I could fixate those sections now?
I tried 10 minutes 4% formaldehyde but it didn’t work.
Thanks.
Hi Yael,
What do you mean by it did not work? Was your tissue clearly not fixed/ too fragile? Did the ensuing stain just not work?
You definitely should be able to do what you said above. 10-20 mins in 4%pfa or ice-cold ethanol/methanol/acetone should be plenty for thin slices like cryosections.
My only thought is that if your tissue is already damaged before (say from dissection, freezing or slicing), fixation will have come too late to help...
Thank you for your answer. I mean that the staining didn't work after just 10 minutes of 4% PFA. probably it wasn't enough time/ formalin concentration to fixate those slices. The tissue is not damaged as far as I can tell. it was sliced and placed on superfrost slides.
I am not sure, though, how to try again. What should I do after thawing the slides? Any thoughts?
I think that you should try a little longer fixation times, rather than stronger fixative solutions. 4% should be plenty...
Unless you think thawing the slices is necessary to help adhere, I'd try going straight from the freezer into the fixative.
Tried 10 min 4% pfa and the staining protocol didn't work. I'll try to do longer fixation. Thanks
Tried 10 min 4% pfa and the staining protocol didn't work. I'll try to do longer fixation. Thanks
https://en.wikipedia.org/wiki/Luxol_fast_blue_stain
http://www.ihcworld.com/_protocols/special_stains/fast_blue.htm
It is easier stain for myelin dont use an oven use a water bath to heat overnight far safer!
When you fix tissue after being fresh frozen, some antibodies will only work with specific fixatives. If 4% PFA didn't work, you can try Acetone or Methanol. Try one slide in each solution and stain and see if you get better results, Most stains work best in 4% PFA, but I have had some histological stains that worked in Acetone but not formalin or in methanol but not acetone. Good luck!
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