Methods and Techniques Discussion => Antigen Retrieval => Topic started by: thomas_leeyl on July 20, 2009, 04:50:49 AM

Title: Problem in AR
Post by: thomas_leeyl on July 20, 2009, 04:50:49 AM
why i can't using the same polyclonal antibody(e.g. flu) in formalin fixed paraffin embedded tissue that it works in frozen section? i have already tried the AR by using pressure cooker with various buffer ( citrate pH 6, EDTA pH 8, target retrieval solution pH9 DAKO). i use 1.5 bar 10 minutes for AR in pressure cooker. it is strange that i use a monoclonal antibody (e.g. H1) for the immunonstaining on the same tissue section, it works perfectly! so i am sure that the AR method is no problem to a certain extent. So, what's the problem is? any other AR method suggested?
Title: Re: Problem in AR
Post by: excalibur on July 20, 2009, 01:33:13 PM
Some antibodies just don't work on paraffin sections, no matter what AR you use.
Title: Re: Problem in AR
Post by: thomas_leeyl on July 21, 2009, 09:48:58 PM
excalibur, then why some antibodies don't work? any hypothesis can explain this? thx!!
Title: Re: Problem in AR
Post by: funk.106 on August 19, 2009, 12:43:55 PM
You could also try an enzymatic treatment after your typical citrate buffer. For example, apply a ProK solution for like, 10 minutes and then continue with blocking or peroxide treatment. I have an antibody that this is the only way I can get it to work on my methacarn-fixed paraffin embedded, but when it does work it's a fantastic stain.