IHC Technical Resources

Protocols, optimization tips,
troubleshooting guides,
and more for IHC.

Troubleshooting guides

Troubleshooting guides

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handbooks for IHC, Western
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  1. 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
  2. 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
  3. ICC/IF (Cell Climbing Slices)

    • Place settled coverslip in culture bottle or perforated plate
    • Take out coverslip after cell growth has reached 60%
    • Wash the coverslip 3X with PBS to remove culture medium
    • Immerse the coverslip (cells face up) into cold acetone or 4% paraformaldehyde or neutral formalin for 10 to 20 min (Close the lid to prevent evaporation)
    • Wash the coverslip 3X with PBS
    • Put the coverslip on filter paper (cells face up)
    • Remove the liquid on the coverslip and allow it to dry for 8-10 hrs
    • To thaw the slice, wash with neutral PBS at room temperature for 10-15 min (The cell climbing slice can be stored in gelatin at -20°C for one week.)

    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.