Surgipath micromount mounting media

Tissue samples were fixed in 10% neutral buffered formalin or 4% paraformaldehyde and embedded in paraffin. Following rehydration and quenching (3% H₂O₂), antigen retrieval was performed by microwaving slides in 10 μM sodium citrate solution for 20 minutes (pH 6). Following the retrieval, slides were blocked using protein blocking solution (DAKO) and then incubated overnight at 4°C with primary antibody (Table 2). For a negative control, non‐specific rabbit IgG (DAKO) was used at the same concentration as primary antibody. Next, sections were incubated with biotinylated secondary antibodies followed by Streptavidin‐HRP. Finally, tissue sections were mounted with Surgipath Micromount^® mounting media (Leica Microsystems Inc.) and examined on an Olympus BX61 Motorized Microscope and MicroSuite^™ system (Olympus America Inc.). The operator was blinded to the treatment groups to minimize operator bias. A minimum of five fields were examined and photographed for each tissue section using Olympus DP72 Microscope Digital Camera (Olympus America Inc.). The number of positively stained immune cells were counted by the blinded operator and recorded to quantify the infiltration of immune cells into the tissue.

Staining was performed by TCP histology laboratory. Sections were baked overnight, deparaffinised, and rehydrated in xylene, 100%, 95%, 80%, and 70% ethanol baths for 5 min each and quenched with 3% H₂O₂ in methanol for 20 min. Next, the tissue sections were washed and stained with a modified Masson’s trichrome histological stain. Verhoff’s Hematoxylin stain was used to stain elastic fibers and nuclei black (6 min of incubation), while Biebrich scarlet-acid fuchsin, with subsequent processing with phosphomolybdic/phosphotungstic acid and aniline blue, was used to stain collagen greenish blue (2–3 min of incubation) and cells in red. The slides were dehydrated following the staining and mounted with Surgipath Micromount mounting media (Leica Microsystems Inc.).