Protocol for Immunohistochemistry Staining of Hematopoetic Cells in Tissues

1. Allow frozen sections to come to room temperature for 1 hour in plastic bag
2. After 1 hour has elapsed open bag and let sit for 30 minutes of further equilibration
3. Fix in acetone (-20^oC) for 10 minutes
4. Wash slides with PBST containing 0.1% BSA
5. Block for 30 minutes at room temperature with 5% normal goat serum (NGS) in PBST/0.1% BSA
6. Wash once with PBST/0.1% BSA.
7. Treat with Avidin 10 minutes at room temperature. (titrate this specificially for your application)
8. Wash once with PBST/0.1% BSA
9. Treat with Biotin 10 minutes at room temperature. (titrate this specificially for your application)
10. Load primary antibody (200ul/slide of ~.2ug/ml or greater-titrate) to paired slides
    • Biotin CD4 in PBS, 0.1% BSA, 1% NGS
    • Biotin CD8 in PBS, 0.1% BSA, 1% NGS
    • Biotin mac-1 in PBS, 0.1% BSA, 1% NGS
    • Biotin GM-CSF in PBS, 0.1% BSA, 1% NGS
11. Incubate from 3 hours to overnight at 4^oC
12. Wash once with PBST
13. Incubate with HRP streptavidin in PBS for 30 minutes at room temperature
14. Wash once with PBST
15. Incubate with DAB with enhancer
    • 7ml DAB substrate
    • 0.385 ml DAB
    • 0.140 ml enhancer
16. Develop for a maximum of 3 minutes. Develop outside of the microprobe holder as it is easier to control the developing
17. Rinse with water and coverslip with aqueous mount

Note: Antibody concentrations here may not hold for all sources. These concentrations should be titrated for each source.