Immpress excel amplified hrp polymer staining kit

The ImmPRESS Excel Amplified HRP Polymer Staining Kit (anti‐rabbit IgG kit: MP‐7601, anti‐mouse IgG kit: MP7602, Vector Laboratories) was employed to amplify signal of potentially weakly expressed antigens. Baking, deparaffinisation, hydration and antigen retrieval was carried out as described above. All further reagents used were provided in the kit. The following day, sections were incubated with BLOXALL blocking solution for 10 min to quench endogenous peroxide activity and subsequently washed in running water for 10 min. Sections were then blocked in 2.5% normal horse serum for 20 min. The block was removed and sections were incubated in primary antibody diluted in 2.5% normal horse serum overnight at 4 °C in a dark moist box. Slides were washed three times in TBST for 5 min and then incubated with Amplifier Antibody for 15 min followed by another two washes in TBST for 5 min. Sections were then incubated with ImmPRESS Excel Reagent for 30 min and washed once in TBST and then in dH₂O for 5 min. Equal volumes of ImmPACT DAB EqV Reagent 1 and 2 were combined and added to sections until they turned brown (10 s – 2 min). Sections were then rinsed in dH₂O followed by running tap water. Slides were counterstained with haematoxylin for 3 s, rinsed with water and dehydrated and mounted as above.