IHC-F protocol

Tissue Preparation

Snap Freezing and OCT Embedding

  • Harvest fresh tissue and place it in a dish filled with ice-cold PBS buffer
  • Wash the tissue thoroughly with PBS to remove blood (Use forceps to remove connective tissues)
  • Cut the tissue into slices of thickness of 3 mm or less
  • Immediately snap freeze the tissue in iso-pentane cooled in dry ice and keep the tissue at -70°C (Do not allow frozen tissue to thaw before cutting)
  • Prior to cryostat sectioning, position the tissue in a mold (which can be simply made by using tin foil) and cover the tissue completely in Optimal Cutting Temperature (OCT) embedding medium
  • Use forceps to take the bottom part of mold into liquid nitrogen for 1 to 2 min (The OCT should change to white)

Cryostat Sectioning

  • Pre-cool a slicer box and detector to -22°C and -24°C, respectively (Ensure the completeness and smoothness of blade)
  • Place the tissue from the mold to the detector where the tissue is fixed
  • Quickly and carefully slice the cryostat sections at 5-10 µm and mount them on gelatin-coated histological slides. Note that:
    • Use coverslip to take sliced tissue
    • Cryostat temperature should be between -15°C and -23°C
    • The sections will curl up if the specimen is too cold
    • The sections will stick to the knife if the specimen is too warm
  • Air dry the sections at room temperature for 30 min to prevent them from falling off the slides during antibody incubations
  • Store the slides at -70°C. Note that:
  • The slides can be stored unfixed for several months at -70°C
  • Frozen tissue samples saved for later analysis should be stored intact
  • Immediately add 50 µL of ice-cold fixation buffer to each tissue section upon removal from the freezer
  • Fix frozen section by immersing it into 4% paraformaldehyde at 2-8°C for 8 min (Or optimally at -20°C for 20 min)
  • Wash the section 3X with PBS and allow it to dry at room temperature for 30 min

Note: This fixation procedure using paraformaldehyde and formalin fixatives may cause autofluorescence in the green spectrum. In this case, you may try fluorophores in the (i) red range or (ii) infrared range if you have an infrared detection system.

Inactivation

  • Mix H2O2 with distilled water (v/v: 1:50)
  • Immerse frozen section or cell climbing slice into the diluted H2O2 at room temperature for 10 min
  • Wash the section 3X distilled water (1 min each)

Antigen Retrieval (Proteolytic Induced Epitope Retrieval: PIER)

  • Dry the frozen sections with filter paper
  • Add compound digestion solution (e.g. Trypsin solution or other enzymatic antigen retrieval solution) to the sections or slices
  • Incubate the sections at room temperature for 3 to 5 min
  • Wash the sections with 3X PBS (5 min each)

Blocking

  • Add 5% BSA blocking solution or normal goat serum to the PIER treated samples
  • Incubate the samples at 37°C for 30 min
  • Discard extra liquid (No washing required)

Primary Antibody Incubation

  • Dilute primary antibody with antibody diluent to the concentration recommended by the antibody manufacturer
  • Add the diluted antibody to the samples and incubate at 37°C for 30 min
  • Wash the samples 2X with PBS (20 min each)

Secondary Antibody Incubation

  • Dilute biotinylated secondary antibody with antibody diluent o the concentration recommended by the antibody manufacturer
  • Add the diluted antibody to the samples and incubate at 37°C for 30 min
  • Wash the samples 2X with PBS (20 min each)

Staining

  • Add Strept-Avidin Biotin Complex (SABC) HRP- or AP-conjugated reagents to the samples
  • Incubate the samples at 37°C for 30 min
  • Wash the samples 3X with PBS (20 min each)
  • Add a suitable amount of DAB reagent to the samples and incubate in dark at room temperature for 10 to 30 min
  • Monitor the tissue staining intensity under a bright-field microscope*
  • Wash the samples 3X to 5X with distilled water
  • Counterstain (if necessary)
    • Add haematoxylin to the sample
    • Dehydrate
    • Immerse the paraffin sections 2X in dimethylbenzene (7 min each)
  • Check the tissue staining intensity under a bright-field microscope

* If the staining background is too high, wash the section 4X with 0.01-0.02% TWEEN 20 PBS and 2X with pure PBS after the SABC reaction and before DAB staining. Then use DAB to stain the samples.