2.2. Immunohistochemistry

Immunohistochemistry was performed using a standard horseradish peroxidase‐conjugated avidin‐biotin complex (ABC‐HRP) method and the signal visualised with diaminobenzidine (DAB) as the chromogen (Vector Laboratories, Peterborough, UK). Sections were deparaffinised, rehydrated to water and endogenous peroxidase activity quenched by placing the sections in 0.3% H₂O₂/methanol for 20 min at room temperature (RT). Sections were subjected to antigen retrieval and following incubation with 1.5% normal serum for 30 min at RT, the sections were incubated with primary antibody, a summary of the primary antibodies and corresponding antigen retrieval methods and conditions used is shown in Table 2. Sections were washed thoroughly in tris‐buffered saline (TBS) and incubated with 0.5% of the relevant biotinylated secondary antibody (Vector Laboratories) for 30 min at RT. After washing in TBS the sections were incubated in ABC solution for 30 min at RT, followed by colour development with DAB. Negative controls were included in every run generated by either the omission of the primary antibody or an isotype control.

Specificity, optimal dilution, antigen retrieval methods and source of antibodies used for immunohistochemistry

Mouse IgG

3

Abbreviations: GFAP, glial fibrillary acidic protein; IBA1, ionised calcium‐binding adaptor molecule 1; MHC‐II, major histocompatibility complex II; MW, microwave; PC, pressure cooker; RT, room temperature; TSC, tri‐sodium citrate buffer.