Immunohistochemistry (IHC) protocol was performed as previously described (11 (link)). Brain tissues collected on day 8 (as described above in Tumor Implantation and Drug Treatment section) were fixed further by immersion in 4% PFA in 1 × PBS at 4°C overnight. The tissues were processed, embedded in paraffin, and 5 mm sections were cut. Every tenth slide spanning the entire forebrain was stained with hematoxylin and eosin (H&E) to identify sections containing the tumor.
IHC was performed after microwave antigen retrieval in a citrate buffer, pH 6.0. The primary antibody and dilution used were as follows: anti-aromatase (1:200; Abcam Biochemicals, ab18995). Biotinylated goat anti-Mouse IgG antibody (Vector Labs) was used as the secondary antibody in conjunction with horseradish peroxidase-conjugatedstreptavidin (Elite ABC, Vector Labs). Aromatase expression was revealed with 3′3′-diaminobenzedine substrate (Vector Labs), counterstained with hematoxylin (Vector Labs), whereas Ki67 and activated caspase-3 were revealed with Vector VIP substrate (Vector Labs), counterstained with methyl green (Vector Labs).
IHC was performed after microwave antigen retrieval in a citrate buffer, pH 6.0. The primary antibody and dilution used were as follows: anti-aromatase (1:200; Abcam Biochemicals, ab18995). Biotinylated goat anti-Mouse IgG antibody (Vector Labs) was used as the secondary antibody in conjunction with horseradish peroxidase-conjugatedstreptavidin (Elite ABC, Vector Labs). Aromatase expression was revealed with 3′3′-diaminobenzedine substrate (Vector Labs), counterstained with hematoxylin (Vector Labs), whereas Ki67 and activated caspase-3 were revealed with Vector VIP substrate (Vector Labs), counterstained with methyl green (Vector Labs).
