Hematoxylin

Recently, TILs, PD-1, and PD-L1 were investigated in this STS cohort [9 (link)]. We used those results to explore the relationship between VISTA and the PD-1/PD-L1 pathway in STS. TILs between tumour cells were counted per high-power field (HPF) (400× magnification, field of view 0.237 mm²) in H&E-stained TMA slides, as routinely carried out by the pathologist.
As described previously [9 (link)], slides were pre-treated with heat and Target Retrieval solution (S1699, Agilent, Santa Clara, CA, USA) before incubation with the monoclonal primary anti-PD-1 mouse antibody (315M; 1:80; Cell Marque, Rocklin, CA, USA) for 60 min at room temperature. The Vectastain Elite ABC HRP Kit (Vector Laboratories, Burlingame, CA, USA) and the chromogen DAB+ (Agilent) were used for detection and Hematoxylin (Vector Laboratories) for counterstaining.
For PD-L1 staining, slides were pre-treated with heat and the Epitope Retrieval Solution pH8 Novocastra (Leica Biosystems, Wetzlar, Germany) before incubation with the monoclonal primary anti-PD-L1 rabbit antibody (E1L3N; 1:50; Cell Signalling Technology) for 60 min at room temperature.
For CD3 staining, a monoclonal antibody raised in rabbit (SP7; 1:150; Zytomed, Berlin, Germany) was employed according to standard procedures. We used the SignalStain Boost IHC Detection Reagent (Cell Signalling Technology) and the chromogen DAB+ (Agilent) for detection.

For assessment of SREBF1 and K130Ac-H2A expressions in the wildtype and Y673/951A xenografts, the tumors were excised, fixed with 10% formalin, paraffin sectioned and embedded. The sections were then rehydrataed and heated at 95 °C for 20 min in 10 mM Sodium Citrate buffer (pH 6.0) for antigen retrieval. The sections were then quenched with hydrogren peroxide briefly to block endogenous peroxidase activity, then permeabilized with 0.4% Triton X-100 in PBS. After blocking with normal horse serum (Vector Laboratories, Burlington, Ontario, Canada) for 30 min, the sections were then incubated with either mouse monoclonal SREBF1 antibody (Santa Cruz Biotechnology, Cat# SC-365513, Clone A4, 1:300 dilution) or rabbit polyclonal K130Ac-H2A antibody (1:300 dilution) at 4 °C overnight. The sections were incubated with biotinylated horse anti-mouse/rabbit IgG for 1 h each. The sections were developed with DAB peroxidase substrate and counterstained with hematoxylin (Vector Laboratories). All slides were then visualized under the microscope and images were captured using EVOS M5000 Imaging System.

To determine the stages of the mouse estrus cycle, the vaginal smear assay was done as previously described [8 (link)]. Vagina was gently washed out with phosphate buffered saline (PBS). The washed vaginal materials were collected and dropped onto Superfrost Plus Stain slides (Thermo Fisher Scientific, Waltham, MA, USA). After drying the slides, vaginal cells were incubated in 50% ethanol, 75% ethanol, and 90% ethanol for 5 min and stained with hematoxylin (Vector Laboratory, Burlingame, CA, USA) and eosin Y (Sigma-Aldrich, St. Louis, MO, USA). After incubating cells in xylene, the slides were covered with Permount Mounting Medium (Thermo Fisher Scientific, Waltham, MA, USA). The stages of the estrus cycle were determined by the cytological features described by Mettus and Rane [4 (link),9 (link),10 (link)]. Each uterus in proestrus, estrus, metestrus, and diestrus stages was collected. One of two uterine horns was processed for paraffin block and the other one was used for RNA and protein isolation.

For H&E analysis, 10-μm sections were cut paraffin blocks. Hematoxylin (Vector Labs, Burlingame, CA, USA) and bluing agent was added. Slides were washed three times with water and once with 70% ethanol (EtOH) for 5 min each. Eosin was added followed by 70, 95, and 100% EtOH, and dipped into xylene. Slides were mounted with Vectashield (Vector Labs) and analyzed with light microscopy. For immunohistochemistry, samples were prepared as described previously (20 (link)) using the following antibodies: Ki67 (1:200; M7249; Dako, Carpinteria, CA, USA), Ki67 (1:100; NB600-1252; Novus Biologicals, Littleton, CO, USA), cleaved-Caspase-3 (1:200; 9661; Cell Signaling Technology, Danvers, MA, USA), MYCN (1:50; 10159-2-AP; Proteintech, Chicago, IL, USA), and CD-31 (1:50; 55024; BD Bioschences, San Diego, CA, USA).

Immunohistochemistry was performed on paraffin-embedded sections. In brief, sections (5 μm) were deparaffinized and rehydrated. Primary antibodies were detected with biotinylated goat anti-mouse IgG (1:2000; Jackson ImmunoResearch Laboratories), biotinylated goat anti-mouse IgM (1:1500), or biotinylated goat anti-rabbit IgG (1:1800) (all from Jackson ImmunoResearch Laboratories) and visualized using an ABC reagent kit (Vector Laboratories), according to the manufacturer’s recommendations. Bright-field images were acquired using a Carl Zeiss Axio Imager M2 microscope, equipped with an Axio Cam MRc5 color camera (Carl Zeiss, Germany). Sections were counterstained with hematoxylin (Vector Laboratories) for nuclear staining. The following antibodies were used for immunostaining: rabbit anti-ATXN1 antibody 11750 (1:700), rabbit anti-oligomer antibody A-11 (1:600), mouse anti-oligomer antibody F11G3 (1:100), and mouse anti-calbindin antibody (1:450).

IHC staining of lung tumor tissue was performed using rabbit anti-human active caveolin-1 antibodies as described earlier [28 (link), 29 (link)] on the mouse model tumors and on 20 de-identified human lung carcinomas provided by the University of Maryland School of Medicine Center for Innovative Biomedical Resources Pathology Biorepository Shared Service, Maryland after an IRB approval from University of Maryland School of Medicine. Briefly, the lung tumors tissues were embedded in paraffin and sectioned at 2-4-μm thickness. Staining was performed using the kit (Abcam, MA, USA) following the manufacturer’s standard protocol (Vector Laboratories, Burlingame, CA). The tissue slides were blocked with 2.5% normal horse serum for 10 min. Samples were then incubated with rabbit anti-human active caveolin-1 antibody (dilution 1:50), for 12 hours at 4°C. Following thorough wash, the tissue slides were incubated with anti-rabbit IgG HRP secondary antibody for 10 minutes. The slides were then stained with 3,3′-diaminobenzidine (DAB) (Vector Laboratories) and counterstained with hematoxylin (Vector Laboratories), dehydrated, treated with xylene, and mounted. All slides were examined, pictures were taken using an Olympus BX41 microscope (Olympus America, Melville, NY).