Hi, I would be thankful if you could help me with some issue in IF staining.
I've fixed some rat brain tissues in PFA (4%) then preserved in sucrose-PBS (30%) for 7-8 months.
Does long storage in sucrose-PBS negatively affect antigens? If so, does antigen retrieval help me to solve this issue? Do you suggest any protocol?
Eagerly awaiting your response
I think storing in sucrose-PBS is a good way not to affect the antigenicity of your samples.
Its good if you try both conditions (antigen retrieval using citrate buffer for 15-20 mins and without) to check which gives a better result.
Hello,
The answer is Yes.
Please see the article by Hidetoshi in J Histochem Cytochm 2003 volume 51, issue 8 (August), pages 995-1003
I never used antigen retrieval on frozen section. I used only on paraffin-embedded sections
I tried with and without antigen retrieval in mice brain. Did not find any difference in IF staining.
Hi,
It will depend on your antigen and primary antibody whether or not antigen retrieval is necessary. According to my experience, it is usually needed when trying to detect fibers or the antibody quality is low. As it damages the tissue a little, I recommend trying without it first. If needed, I usually incubate brain slices for 15 minutes at RT in HCl 2N, then wash 3 times with PBS 0.1M, and then start with the blocking step.
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