Mouse Ab on Mouse Tissue: Blocking Problem - (Sep/10/2007 )

Hi All,
I'm sure this question/problem has been on this forum before, but I couldn't find a reference so here goes.

I am using a mouse monoclonal on mouse tissue, and have tried using a goat anti mouse IgG incubation step before the primary antibody step to prohibit binding of the secondary antibody, goat anti mouse IgG (Fab2)-Alexa Fluor, to the mouse tissue. However, I am still getting quite a high level on binding of the secondary antibody to the mouse tissue.
So far I have used the goat anti mouse IgG at 1/100 for 2 hours at RT then incubated with the primary antibody o/n at 4C, then incubated with the secondary antibody 35min at RT. I've got no primary as a negative control and tissue sontrols for the primary antibody.
I know the best solution is probably to use a MOM- mouse on mouse kit, however we have the above reagents available and want to see some results before thinking about spending more money.
Does anyone have any suggestions for me, or experienced the same problems.

Cheers.

Conjugate your first Ab to some dyes?

I've recently used a mouse primary antibody on a mouse tissue, recognized with goat anti-mouse Alexa Fluor antibody.

To avoid unspecific binding of the sec Ab on tissue I just add a "blocking step" (1 hour RT) after the overnight incubation with the primary Ab.

I was suspicious when my colleague gave me this protocol, but that's a fact: it works.

so, in summary:

-fixation
-washes
-blocking (BSA5% PBS Tween 20 0.1%) 1h RT
-prim Ab 4°C o.n. (BSA1% PBS Tween 20 0.1%)
-washes
-blocking (BSA5% PBS Tween 20 0.1%) 1h RT
-sec Ab 90min RT (BSA1% PBS Tween 20 0.1%)

I hope this may help.

Hi All,
I'm sure this question/problem has been on this forum before, but I couldn't find a reference so here goes.

I am using a mouse monoclonal on mouse tissue, and have tried using a goat anti mouse IgG incubation step before the primary antibody step to prohibit binding of the secondary antibody, goat anti mouse IgG (Fab2)-Alexa Fluor, to the mouse tissue. However, I am still getting quite a high level on binding of the secondary antibody to the mouse tissue.
So far I have used the goat anti mouse IgG at 1/100 for 2 hours at RT then incubated with the primary antibody o/n at 4C, then incubated with the secondary antibody 35min at RT. I've got no primary as a negative control and tissue sontrols for the primary antibody.
I know the best solution is probably to use a MOM- mouse on mouse kit, however we have the above reagents available and want to see some results before thinking about spending more money.
Does anyone have any suggestions for me, or experienced the same problems.

Cheers.