[Histonet] Mouse abs on mouse tissue method

[Emily Sours]

This doesn't really help solve your problem, but I have a question.  Why
are you using two secondary antibodies?
Why not just use the conjugated one?

Emily
(starting Friday off right by eating candy for breakfast.  I love Fridays!!)


The whole point of this country is if you want to eat garbage, balloon up
to 600 pounds and die of a heart attack at 43, you can! You are free to do
so. To me, that’s beautiful.
--Ron Swanson



On Fri, Mar 23, 2012 at 8:34 AM, James S. <sonya.martin <@t> soton.ac.uk> wrote:

> 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
>
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