[Histonet] Mouse abs on mouse tissue method

[James S.]

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