Immunofluorescent Staining Of ECM Proteins In Lung Tissue

Antigen retrieval by Proteinase K digestion.
  1. Incubate paraffin imbedded and sectioned tissue slides @50oC for about 20’.
  2. Xylene 2 X 10’
  3. 100% EtOH 2 X 3’
  4. 95% EtOH 2 X 3’
  5. 79% EtOH 5’
  6. Wash in running water 5’
  7. Warm up slides for 5’ in pre-warmed dH2O at 37o
  8. Antigen retrieval by Proteinase K digestion:
  9. Incubate slides 5’ in pre-warmed (37oC) solution of Pk 20 µg/ml in TE (100µl of 10 mg/ml stock in 50ml TE)
  10. Wash 3 X 5’ with dH2O
  11. 45’ 100 mM CuSO4 (quenching of auto-florescence)
  12. Wash 3 X 5’ in TBS / 1% BSA. [TBS: 150 mM NaCl, 50 mM Tris, pH7.6]
  13. Blocking in 10% serum/ TBS/ 1% BSA, at least 1h/RT/humid chamber. Use »200µl of blocking sol/slide
  14. 1st Ab in 10% serum /TBS / 1% BSA. ON at 4oC in humid chamber