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HOME > Protocols > Histology > Staining protocols > Protocol for Immunohistochemistry Staining of Hematopoetic Cells in Tissues

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 (-20oC) 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 4oC
  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.


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