IHC/ICC/IF
Sample Preparation

Snap Freezing and OCT Embedding

1. Harvest fresh tissue and place it in a dish filled with ice-cold PBS buffer
2. Wash the tissue thoroughly with PBS to remove blood (Use forceps to remove connective tissues)
3. Cut the tissue into slices of thickness of 3 mm or less
4. 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)
5. 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
6. 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

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

1. Place settled coverslip in culture bottle or perforated plate
2. Take out coverslip after cell growth has reached 60%
3. Wash the coverslip 3X with PBS to remove culture medium
4. 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)
5. Wash the coverslip 3X with PBS
6. Put the coverslip on filter paper (cells face up)
7. Remove the liquid on the coverslip and allow it to dry for 8-10 hrs
8. 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.

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