Hi, everyone, I am working on immunostaining of bone marrow and growth plates of mice. And I have to use an mouse monoclonal Ab to do this staining. However, with only secondary goat anti-mouse Ab, it showed very strong background within bone marrow. I then tried using Goat Fab anti-mouse IgG(H+L) to block endogenous IgG (40ug/ml in PBS for 3 hours) before primary Ab. Indeed, the Fab blocking reduced background to about 20%, but the fluorescent signals of my protein also significantly decreased compared with no Fab blocking group. Is there any problems with my protocol? or any tips about mouse on mouse IF staining?
Here is my protocol for endogenous IgG blocking:
1. After antigen retrieval, rinse slides in PBS 3 times.
2. 1% Triton-x-100 for 30 min.
3. Rinse in PBS 3 times.
4. Serum blocking for 60 min at RT.
5. Rinse in PBS 3 times.
6. Dilute goat Fab anti-mouse in PBS (without serum) at 40ug/ml, incubate for 3 hours at RT.
7. Rinse in PBS 3 times.
8. Primary Ab at 4 degree O/N.
Jifan, in indirect immunodetection systems, avoid secondaries against the sample species at all cost. If this should ever work, optimizing will take more effort and cost than switching to antibodies from another species and if it should apparently work, you're still risking your results being questioned by serious reviewers.
For a first shot, you could try to directly label your primary antibody with a reactive fluorescent dye (FITC, TRITC, Alexa NHS esters, etc) or Biotin-NHS ester (use streptavidin-HRP for detection), so that you don't need any secondary at all.
Perhaps you could adapt the protocol we have recently published for staining the mouse cochlea: Tackling the Mouse‐on‐Mouse Problem in Cochlear Immunofluorescence: A Simple Double‐Blocking Protocol for Immunofluorescent Labeling of Murine Cochlear Sections with Primary Mouse Antibodies https://doi.org/10.1002/cpmo.84
Dear Researcher,
Sharing with you a double‐blocking protocol for Immunofluorescent labeling of murine cochlear sections with primary Mouse Antibodies
https://doi.org/10.1002/cpmo.84