IHC (Paraffin Sections)

Paraformaldehyde Cooling and Dehydration

  • 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
  • Immerse the slices in 4% paraformaldehyde at room temperature for 8 min
  • Immerse the slices in 4% paraformaldehyde (pre-cool at 4°C) for 6 to 7 hrs. The paraformaldehyde volume should be 20X greater than the tissue volume by weight
  • Wash the tissue 3X with PBS (1 min each)
  • Dehydrate the tissue by immersing the tissue sequentially as follows:
    • 1X into 80% ethanol (1 hr at 4°C)
    • 1X into 90% ethanol (1 hr at 4°C)
    • 3X into 95% ethanol (1 hr each at 4°C)
    • 3X into 100% ethanol (1 hr each at 4°C)
    • 3X into dimethylbenzene (0.5 hr each at room temperature)

Liquid Paraffin Section

  • Prepare the first portion of liquid paraffin in a suitable bath and allow the paraffin to reach and maintain at 60°C
  • Immerse the tissue 2X into the paraffin bath (2 hrs each)
  • Prepare the second portion of liquid paraffin in a suitable bath and allow the paraffin to reach and maintain at 60°C
  • Pour the second portion of paraffin into a mold
  • Quickly transport the tissue from the paraffin bath to the mold with paraffin
  • Incubate the tissue at room temperature until it coagulates
  • Store the tissue at 4°C

Section Slicing and Incubation

  • Secure the paraffin section on slicer
  • Slice one to two pieces of section to adjust the slicer so that the section and blade are parallel
  • Slice the remaining section carefully with ~5 µm thick
  • Incubate the sliced section in 40 to 50°C water to unfold
  • Mount the tissue section onto Poly-Lysine or APES coated glass slides
  • Incubate the slides overnight at 37°C