Occasionally we observe lack of staining in the inner part of a skin section processed with indirect immunofluorescence. Can be due to incomplete removal of the cryoprotectan during the wash? Any suggestion?
Could be. Here I recommand intensive washing steps before you start your immuno. 3 x 10 min PBS or TBS, leave the sections in the last buffer step over night. It could be also the case that the polyvenyl-pyrrolidone Ethylenglycol mixture makes problems to wash out. Should this be the case then I recommand to change the cryprotection solution. Use 30% sucrose in PBS and Glycerol equal parts. This solution makes no problems neither in immunohistochemistry with DAB staining nor in immunofluorescnence.
Ute Neubacher Thank you very much for your comments and your advices. I will try them.
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