Immunohistochemistry or IHC refers to the process of localizing proteins in cells of a tissue section exploiting the principle of antibodies binding specifically to antigens in biological tissue.

The Molecular Pathology Core can provide IHC staining, please review the following information or contact Kelly.

1. Provide (or order) antibody(ies) of choice - to label tissues (primary antibody).

2. Select and retrieve “positive control tissue(s)” - including species type to demonstrate your primary antibody.

3. Prepare tissues for frozen and/or paraffin sectioning onto slides (fixation).

Frozen sections: Snap freeze tissues in OCT in tinfoil (in a mixture of liquid nitrogen and isopentane)
and store at -80°C (freezer). Cryo-protect if necessary.

Paraffin sections: Place tissues in a tissue cassette in either 4% paraformaldehyde (4 hours then transfer to 70% ethanol), or place in formalin; Paraffin process, and embed tissue.

4. Tissue sectioning - onto positively charged slides from frozen or paraffin blocks.

5. Stain sections of control tissues - with primary antibody using variable dilutions to determine optimum concentration.

6. View sections under microscope – determine if control sections and dilutions provide representative positive staining.

• Yes – go on to step 7
• No – retrieve different representative tissue samples and/or primary antibody, and consider different labeling systems or pretreatments.

Part B: Immunohistochemistry Staining (Core Laboratory)

7. Fix, process (if paraffin embedded), and cut “test tissues” - onto positively charged slides.

8. Stain (test tissues and positive control tissues) - using the immunohistochemistry protocol of choice. Determine if staining is representative. Adjust and repeat as needed.*

Part C: Interpretation (Requisitioner and/or Core Laboratory)

9. Microscopy and imaging of tissue sections.