immunohistochemistry high backgroungd staining - (Aug/01/2008 )

hi friends,
for last one month i am feeling like washermen... washing-1 Ab-washing-2Ab-washing only that is going on..
I am doing immunofluorescence( FITC) staining of spleen sections of mouse..
sections r 20um thick (cryosections)...
for primary Ab i m using 1:250 dilution, for sec. (anti mouse Ab) tried different dilutions 1:200 - 1 :10000..
for blocking using 3% horse serum...
washing with 1X PBS.. but again n again i am gatting very high background staining....


send me reply soon to again make me research scholar from washermen....

waiting for reply

gnp

Sounds like your blocking isn't very effective. The best block is the serum of the species in which your secondary was produced. If you have a goat anti-mouse secondary, block in goat serum. I minimally block in 5% BSA or goat serum. For very high background I do a primary block of BSA, primary antibody at highest dilution, wash, reblock in 5% goat serum, secondary, wash. Are you sure 1:250 is the best dilution for your primary? You should run a dilution series to determine the best. Keep your secondary at 1:10000 and determine the best primary dilution. Most background comes from the secondary so the higher this dilution the better. Another blocking idea, I've seen people swear by cold fish skin gelatin. I bought it and didn't see much of a difference for my antibodies but you never know what yours will prefer. How long are you blocking? Over-night will never hurt. Next, wash conditions. You can add a bit of TX100 (0.1-0.5%) to try to disrupt the non-specific. I've had this greatly help reduce background. How long are your washes? How often are you changing buffer? Again, I have some antibodies I wash overnight in a high volume of buffer.

Great topic, I have the same problem...
I'm just starting to set up IF staining w/ different primary antibodies on uninfected spleens (cryo)sections of mouse.
I finished a run w/ different dilutions, primary ab. Different secundary antibodie, different counterstaining, but they all look the same!..
Everything seems to be stained, (not frustrating at all you know!)
Anyway, thanks for the good ideas and keep them coming!

Blocking with serum from same species as secondary ab will not help. Your problem is using mouse primary antibody on mouse tissue. Any anti-mouse secondary will bind to endogenous mouse Ig in your tissue. (Mouse spleen is loaded with mouse Ig!) Avoid primary antibodies raised in mouse when working on mouse tissue, or use designated MOM (Mouse on Mouse) Kit.

ImmUnoUno is right. you absolutely need a MOM kit (vector labs makes a good one for IF). I don't get any background when I use this kit. Also could try H2O2 and CAS block instead of the serum block

hths

I don't have much experience in immunohistochem side but in immunoassay side it seems you have the same problems: heterophile antibodies.

Ab in the mouse tissue against ab1 and ab2
Also ab1 may non-specifically rx with ab in mouse tissue AND ab 2
Finally ab2 may non-specifically rx with ab 1 and ab in mouse tissue.

These are all possible. Your primary ab and secondary abs should be specific. Non-specific sera diluted or otherwise may NOT resolve your problem. Best case is purified non-specific ab same species as ab 1, ab 2 and of course mouse. You wish to have your ab bind to target and block non-specifics by pre-exposing them to non-specific ab (same rational as using the sera...but this time you give them much higher concentration IgG as blocking agent).

Note: non-specific titers can be extrodinarily high.

Can you pre-incubate/pre-dilute your abs and mouse tissue with non-specific ab prior to testing??