Immunohistochemical Analysis of Tissue Sections

Briefly, deparaffinization and rehydration of 5 μm sections were performed as follows: 1. three incubations for 3 min in xylene; 2. three incubations for 2 min in 100% ethanol; 3. 2 min each in 95, 80, and 70% ethanol; 4. 5 min in 1× TBS (Tris-Buffered Saline). All chemicals, BSA, and buffers were purchased from Fisher Scientific, Pittsburgh, PA, USA. Antigen retrieval was performed using a citrate buffer, pH 8.0 at 98C, for 20 min, as previously reported [32 (link)]. Sections were subsequently blocked using 5% Bovine Serum Albumin (BSA), 1× TBS solution. Sections were stained with anti-Ki67-AlexaFluro-555 conjugated antibody (BD Biosciences, Franklin Lakes, NJ, USA), anti-cytokeratin 8 [33 (link)] (1:100 dilution, TROMA-I was deposited to the DSHB (Developmental Studies Hybridoma Bank) by Drs. Brulet, P. & Kemler, R.), and anti-cytokeratin5 (Poly19055, BioLegend San Diego, CA, USA), dilution 1:500, and incubated overnight, as previously reported [34 (link)]. Secondary antibodies that were diluted, 1:10,000, included AlexaFluor-488, 555, and 647 (ThermoFisher Scientific, Waltham, MA, USA). The sections were mounted with ProLong Gold DAPI-containing media (ThermoFisher Scientific, Waltham, MA, USA). Images were captured using an Olympus VS120 Slide Scanner microscope with 10× U Plan S Apo/0.75 NA objective (Olympus Lifescience, Center Valley, PA, USA).
For H&E, staining slides post deparaffinization and rehydration were incubated with hematoxylin (Fisher Scientific, Pittsburgh, PA, USA) for 45 s, rinsed with water for 30 s, followed by Eosin for 1 min, rinsed and dehydrated in 95% ethanol, followed by three incubations for 1 min in 100% ethanol and three incubations for 1 min in xylene. Slides were mounted using Richard-Allan Scientific Mounting Medium (ThermoFisher Scientific, Waltham, MA, USA). Samples were evaluated by an experienced pathologist blinded to the identity of the slides.