[Histonet] IF staining on peritoneal macrophages

[Andrea T. Hooper]

Dear Mauricio,
 
The two antibodies you chose were NOT cross adsorbed against mouse. This is critical for successful staining of mouse tissue with rat antibodies. These are the two alternate catalog #s of the ones I would recommend if FITC is your thing:
 
712-095-153 (whole IgG FITC conjugated)
712-096-153  (Fab'2 FITC conjugated)
 
Although the important part to reduce background and increase sensitivity is the antibody itself, the choice of fluorophore can also play a huge role in your success. There are now many much better fluorophores than FITC and I suggest you play around with some of those. 
 
Firstly are you fixed on using the "green channel" (ie FITC) vs the "red channel" (ie rhoadmine)? I personally like CY3 (red) the best for brightness of the Jackson IR fluorophores. CY2 is also great. They also have a new series called DyLights which are reported to be great. I have played with them a little bit with some success and some failures.
 
Ok, so assuming you want to stick with the green channel (I suggest you use CY2 then) these are the two simple choices for you  (the second one is a F(ab')2 and one is a whole IgG - 99/100 the whole IgG will be fine for you and is what I use for staining hematopoietic tissue ...):
 
712-225-153 (whole IgG CY2 conjugated)
712-226-153 (Fab'2 CY2 conjugated)
 
Another option for boosting signal is to use a biotinylated antibody and then use streptavidin conjugated to your fluorophore of choice. Then if you want to use the Alexa488s you can get streptavidin-Alexa488 from Invitrogen.
 
712-065-153 (whole IgG biotinylated)
 
In my lab, I use both the CY2/CY3 conjugates as well as the biotin conjugates routinely. I rarely if ever use the F(ab')2 antibodies, it's normally overkill.
 
Let me know if you need anything else,
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.