[Histonet] RE: mouse abs on mouse tissue

[Reynolds,Donna M]

I am not familiar with what IHC world has on this but we have used this technique for a number of years. The mouse fragment we use is from Jackson  cat #115-007-003. We dilute it 1:10 in our protein blocking solution. It works better with DAB than with Fluorescence. We have also found that to get optimum blocking we need to incubate overnight at 4 degrees. This is more critical with some tissues and antibodies than with others. So we just routinely do it overnight if at all possible. If not we incubate for as long as possible. We usually get at least 98% blocking with this method.
With fluorescence we us a 10% cold water fish gelatin (from Electron Microscopy) for our protein blocking solution. This really has cleaned up all our fluorescent labeling. With Dab our normal serum blocking is ok. You didn't say the dilution your were using for the Dylight. You may need to titer it out a little further. I have just recently learned that Jackson is also carrying the mouse Isotype specific antibodies with fluorescent dyes. We have always used the HRP isotype specific antibodies to help clean up our mouse abs on mouse tissue. I have ordered but not tried the fluorescent ones I am expecting good results. 

Donna Reynolds, ASCP
Chief Histology Lab, 
U.T. M.D. Anderson Cancer Center, Houston TX
Department Cancer Biology
713-792-8106 
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Message: 20
Date: Fri, 23 Mar 2012 12:34:01 +0000
From: "James S." <sonya.martin <@t> soton.ac.uk>
Subject: [Histonet] Mouse abs on mouse tissue method
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I've been trying out the method from IHC world to use mouse primary plus anti-mouse secondary abs on mouse tissue.

Protocol;

Fresh frozen sections (mouse spleen) cut 8um and air dried
Fixed acetone 10min
Wash PBS
Block with 2.5% normal goat serum in PBS/Tw, 30min
Block with unconjugated Fab fragment goat anti-mouse IgG (Jackson Immuno, diluted 1/10 in PBS/Tw), 2hrs room temp
Wash PBS/Tw
Seconary ab - DyLight488 conjugated Fab fragment goat anti-mouse IgG (Jackson Immuno, 1/500 PBS/Tw), 20min room temp
Wash PBS/Tw
Mount

Blocking with unconjugated Fab did decrease background if I used an IgG goat anti-mouse secondary but the background was actually increased when I used the Fab goat anti-mouse!!

Any suggestions?

Thanks
Sonya