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HOME > Protocols > Histology and IHC > Staining protocols > Protocol for Immunohistochemistry Staining of CD80 and CD86 in Tissues

Protocol for Immunohistochemistry Staining of CD80 and CD86 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-anti-mouse CD80
    • Biotin-anti-mouse CD86
    • 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-D-avidin in 0.1M soidum bicarbonate and 0.15M NaCl (pH 8.5) 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 1 minute. 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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