I'm doing immunofluorescence imaging on frozen sections of guinea pig and pig cochleae. The antibodies that I'm using are anti-CTGF to stain type 4 fibrocytes and anti-AQP1 to stain type 3 fibrocytes. The disadvantage of those antibodies that I've encountered is that they give a lot of background staining.
I want to try an antigen retrieval (AR) step before starting the immunofluorescence protocol that I'm already using to see if it would make any difference. The problem is that heating frozen sections in antigen retrieval as with paraffin sections will probably destroy my samples.
Is there an AR protocol that is good for frozen sections without any heating? I've already found an AR protocol that uses 1% SDS in PBS. Are there others using e.g. citrate buffer?
Thanks in advance for your help!
Hello Lana Biot
You may be knowing that for frozen sections, antigen retrieval is not required as the fixation time with aldehydes is very short (10–30 minutes) and doesn’t allow the formation of cross-links. The purpose of antigen retrieval is to break the aldehyde bonds that form when you fix the tissue. Nevertheless, you may try the antigen retrieval protocol mentioned in the attached paper.
Preprint Transcription factor MafB is a marker of tumor-associated ma...
Protocol in brief:
Antigens were retrieved by autoclaving at 105 degree C for 1 min only in TE buffer, pH 9.0, followed by a gradual decrease of temperature to room temperature.
Hope this will work!
Best.
Hi Lana,
You can try different things to reduce your background. We work with frozen sections of pigs. We wash with 1x TBS/0.1% Tween (All washing steps: 4x 10 min)...
1. antibodies in TBS/0.1 %Tween/1%BSA --> centrifuged 5 min, 10 000 RPM ! Before adding...
Incubate over night at 4 degree... this will also reduce unspecific binding...
2. Antibodies in TBS/0.1 % tween/ 5% porcine serum--> centrifuged 90 sec, 10 000 rpm ...1h, RT...
I hope this will work...
Best regards,
Constanze
I never used antigen retrieval on frozen sections in rat and mice. But additional and interesting information from Constanze Nossol of centrifugation of antibodies to remove non-specific background. Thanks, Constanze