[Histonet] IF staining on peritoneal macrophages

[Andrea T. Hooper]

Sorry for the second email, I didn't address all your questions ...
 
The antibody I use from Serotec is MCA497, works very well and many people on Histonet have claimed the same over the years.
 
I am not familiar with the secondary you use from Serotec. It says it's goat anti-rat IgG, mouse adsorbed. So it should be ok? But again FITC really isn't a great fluorophore and I tend not to use anything except Jackson IR reagents.

I don't understand why they would suggest to use mouse serum, especially since it's mouse adsorbed?  Unless you are trying to block Fc receptors - again, probably overkill. I have stained mouse hematopoietic tissue for years with rat antibodies and secondaries from Jackson and have never had this problem. I think this mouse serum step will just add background. Don't do it. Usually one is supposed to dilute their secondaries in the serum that the secondary is made in. Therefore in your case, I would use goat IgG. Block in your favorite blocking recipe ... For the antibodies I recommended to you, I use 10% donkey serum with 5% BSA (make sure it's gamma globulin free) in buffer with some detergent. Incubate primaries in 0.2X concentration of your block.
 
If you want to share your protocol, maybe I can give you some further tips?
 
Let me know if I can help further,
Andrea
 

--- On Wed, 3/3/10, Mauricio Avigdor <bitesizellama <@t> gmail.com> wrote:


From: Mauricio Avigdor <bitesizellama <@t> gmail.com>
Subject: Re: [Histonet] IF staining on peritoneal macrophages
To: "Andrea T. Hooper" <andreahooper <@t> rocketmail.com>, histonet <@t> lists.utsouthwestern.edu
Date: Wednesday, March 3, 2010, 8:18 PM



Hi Andrea,
 
Jackson lists a couple of FITC-conjugated donkey anti-rat secondaries:

712-095-150 - Whole Donkey Anti-Rat IgG (H+L).
 




712-096-150

- F(ab')2 fragment Donkey Anti-Rat IgG (H+L).
 
Do you recognize which is the one you have ben using.
 
I have been using Serotec's STAR80F antibody. They suggest using 1:10 Normal Mouse Serum in PBS to make the necessary dilutions. Is this something you do with the Jackson antibodies as well? I've never heard of this technique before.