Histopathological Analysis of Tumor Samples

Tissue samples from mice bearing tumors from Black and White PCa patients post treatments with GemHCl and 4NSG-SLN were excised and immediately washed with PBS, fixed (10% buffered formalin) for 24 h, and transferred to 70% ethanol for histopathological analysis. Histology was performed by HistoWiz Inc. (Histowiz.com) according to manufacturer protocol. Samples were embedded in paraffin, and 4 μm sections were prepared [7 (link), 40 (link), 41 (link)]. Immunohistochemistry was performed on a Bond Rx autostainer (Leica Biosystems) with enzyme treatment (1:1000) using standard protocols (https://home.histowiz.com/faq/). Samples were incubated overnight with primary antibodies (Antibodies used were rat monoclonal F4/80 primary antibody (eBioscience, 14–4801, and 1:200), and immunohistochemical staining was done using rabbit anti-rat secondary (Vector Labs, 1:100) [7 (link), 40 (link), 41 (link)]. Bond Polymer Refine Detection was used per the manufacturer's protocol (Leica Biosystems). After staining, sections were dehydrated and filmed, coverslipped using a Tissue-Tek Prisma coverslip (Sakura). Whole slide scanning (40x) was performed on an Aperio AT2 (Leica Biosystems).