[Histonet] Mouse CD45 staining of bone marrow

[Adam .]

Hi all,

I have been attempting to do mouse CD45 immunofluorescence on frozen, fixed
decalcified mouse sections and have an odd problem. I am using the
monoclonal rat anti-mouse CD45 (clone 30F-11 from BD) and get very nice
staining along the cell surface of many cells. However, only about 20% of
the nucleated cells stain with CD45.

By FACS, > 90% of the cells stain with CD45 using the same clone. To be
fair, that is with RBC lysis, but the RBCs should not be nucleated anyhow.
The antibody comes at 62.5 ug / mL, and I am using the antibody at a 1:1000
dilution in TBS-T. I found that at the recommended1:20 titer, nothing
stained above background. I only began to get staining at < 1:100,
suggesting to me there may have been some steric hindrance going on.
Although this clone is supposed to work on FFPE sections, I have been unable
to get any staining at all using even with aggressive HIER.

I was wondering if anyone has IHC or IF photos of CD45 staining in mouse
bone marrow so I know what it should look like. Any suggestions are welcome.

Thanks,
Adam

The specifics:
1) Fix the bones overnight in 4% PFA at 4C
2) Decalcify in EDTA for 3 days at 4C
3) Cryoprotect in 30% sucrose overnight at 4C
4) Embed in OCT using liquid nitrogen cooled 2-methylbutane
5) Section using Cryojane tape transfer system. I have also tried this with
a regular cryotome with similar results.
6) Block in 10% donkey serum, then avidin and biotin
7) Incubate with primary antibody overnight at 4C. Wash.
8) Incubate with biotinylated donkey anti-rat F(ab)2 (1 ug / mL) at room
temperature for 1 hr. Wash.
9) Incubate with strepavidin Dylight 594 (1 ug / mL) at room temperature for
1 hr. Wash.
10) Mount with Prolong Gold anti-fade with DAPI