IHC (Frozen Sections)

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