IHC Protocol for Bone

Date:

Purpose of Experiment:

 

Slides:

 

Protocol:

  • Heat slides at 60 degrees C for 30 minutes.
  • Deparaffinize and Rehydrate
  • Xylene 3 X 5 minutes
  • 100 % EtOH 3 X 3 minutes
  • 95 % EtOH 2 X 3 minutes
  • 70 % EtOH 2 X 3 minutes
  • 50 % EtOH 2 X 3 minutes
  • PBS 1 X 3 minutes
  • Block endogenous peroxidase activity
  • 3.3 ml Hydrogen Peroxide + 96.7 ml MeOH.  Incubate at room temperature for 30 minutes.
  • Wash 1 X PBS 3 minutes
  • Block with TBST + 20% Aquablock for 30 minutes.
  • Add primary antibodies diluted in TBST + 5% BSA.  Incubate O/N at 4 degrees C.
  • Antibodies:
Check if used Primary antibody name Company and catalog information
Rat anti-endomucin (1:50) Santa Cruz; SC-65495
Goat anti-Lyve-1 (1:250) R&D Systems; AF2125
Hamster anti-podoplanin (1:1000) Abcam; AB11936
Chicken anti-GFP (1:4000) Abcam; ab13970

 

    1. Wash 3 X 3 minutes with TBST.
    2. Dilute secondary antibodies in TBST + 5% BSA.  Incubate at room temperature for 60 minutes.
  • Antibodies:  
Check if used Secondary antibodies (All used at 1:500)
Jackson ImmunoResearch HRP conjugated donkey anti-rat
Jackson ImmunoResearch HRP conjugated donkey anti-goat
Jackson ImmunoResearch HRP conjugated donkey anti-rabbit
Jackson ImmunoResearch HRP conjugated donkey anti-hamster
Jackson ImmunoResearch HRP conjugated donkey anti-chicken

 

  1. Wash 3 X 3 minutes with TBST.
  2. Add DAB reagent.  Develop for _____ minutes.
  3. 1 X 3 minutes in water.
  4. 5 dips in Hematoxylin.
  5. Wash with tap water.
  6. Dehydrate slides (15 dips each)
    1. 2 X 50% EtOH
    2. 2 X 70% EtOH
    3. 2 X 95% EtOH
    4. 3 X 100% EtOH
    5. 3 X Xylene
  7. Add cytoseal and coverslips.