Immunohistochemistry: F4/80

Protocol at room temperature unless otherwise stated.

Positive control section: bowel

  • Dewax
    • Histaline, 5min
    • Histaline, 5min
    • 100% ETOH, 2min
    • 100% ETOH, 2min
    • 70% ETOH, 2min
    • Running tap water, 5min
    • Transfer to distilled water
  • Wash three times in PBS (approximately 2mins each)
  • Cover slides with 3% H2O2 (6% H2O2 Diluted 1:1 in PBS) - 10mins
  • Wash three times in PBS (approximately 2mins each)
  • Digest with pepsin [0.1% (1mg/mL) pepsin in 3% acetic acid] - 10mins, 37 degrees
  • Wash three times in phosphate buffered saline (PBS) (approximately 2mins each)
  • Cover Slides (2mins) with protein blocking agent (PBA) (diluted 1:1 in dH2O) - 10mins
  • Wash three times in PBS (approximately 2mins each)
  • Cover slides with primary antibody; rat anti-mouse F4/80 - 1 hour
    • (Serotec)
    • Dilute to 10ug/mL (1:100) in 1% BSA/PBS
    • Negative control; rat IgG2ak (Sigma #15131)
    • Dilute to 10ug/mL in 1% BSA/PBS
  • Wash three times in PBS (approximately 2mins each)
  • Cover slides with secondary antibody - 1 hour
    • Biotinylated goat anti-rat IgG (Chemicon AP183beta)
    • Dilute 1:200 in 1% BSA/PBS
  • Wash three times in PBS (Approximately 2mins each)
  • Cover slides with strepavidin-peroxidase conjugate - 15mins
    • Use neat from bottle (LSAB-2, Dako#K1016)
  • Wash three times in PBS (Approximately 2mins each)
  • Cover slides with DAB chromagen - 5mins
    • (DAB peroxidase substrate kit, Sigma #D4168)
    • To 1mL dH2O, add 1 tablet of DAB and 1 tablet of Urea and vortex
  • Wash three times in dH2O (Approximately 2mins each)
  • Mount with Dako Faramount aqueous mounting medium and leave slides overnight to dry