[Histonet] anti-GFP staining rabbit polyclonal A11122
koellingr <@t> comcast.net
koellingr <@t> comcast.net
Mon Feb 27 21:12:45 CST 2012
I agree with Paula Pierce, have used the same anti- GFP antibody on some projects but at 1:500-1:1000 area and with a much longer 8-15 minute retrieval in citrate. Also, I know you know to be aware of "how much" the mice are transfected . In my transfected mice (NOT GFP and in a former life) we might transfect 4 different times and get 4 different efficiencies and 4 different "levels" of expression in 4 different lines. And of course then in your case "the variation with the type of GFP " you are transfecting with might ultimately be seen at varying IHC levels since all GFP antibodies don't see all GFP variants with the same efficiency.
Something I did to help ease the pain of working up a stain when working up lacZ (not GFP ) was to use a commercially available Tie2/ LacZ transgenic mouse. With that Tie2 promoter driving LacZ expression only in endothelial cells, you had neg tissue (equivalent to wt) and pos endothelial cells (your transgenic target) right in the very same section. It proved to be an exquisitely sensitive control when doing beta-gal studies. Although I've never used it I understand there is now a Tie2/ GFP transgenic available which should help optimize staining protocols as the expression levels are constant and well characterized and you need only one mouse. Don't know what other gene or transgene target you are after but any given transgene does not incorporate equally all the time as you know. Thats my take on this.
----- Original Message -----
From: "Marina Pils " <Marina. Pils @helmholtz-hzi. de >
To: " histonet @lists. utsouthwestern . edu " < histonet @lists. utsouthwestern . edu >
Sent: Monday, February 27, 2012 4:05:56 AM
Subject: [ Histonet ] anti- GFP staining rabbit polyclonal A11122
We are trying to establish an anti- GFP staining with rabbit polyclonal A11122 from Invitrogen .
We are using formalin fixed, paraffin embedded mouse tissue and want to distinguish between a GFP transgenic mouse strain and a wt mouse.
The problem is, we get a strong background in the wt, especially in spleen and lymphnodes , while the staining looks fine in liver.
We dilute the antibody 1:400 and use 2 min antigen retrieval in Citrate buffer.
Can anyone recommend a protocol? Or any other antibody that is working better?
Thanks a lot for your help,
Marina Pils , DVM , PhD
Animal experimental unit
Helmholtz Centre for Infection Research
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