Hematoxylin 2

Cell line and tissue IHC samples were processed on a Roche Tissue Diagnostics BenchMark ULTRA automated staining platform using OptiView DAB detection chemistry (VMSI Cat# 760–700) with Hematoxylin II (790–2208) and Bluing (760–2037) counterstain. Bulk staining reagents were from Roche Tissue Diagnostics. The SP464/17B4 assay conditions consisted of 1 h of heat-based epitope retrieval on 4 µm thick sections of FFPE hepatocellular carcinoma specimens. Rabbit SP464 antibody was tested at a concentration of 49 nM (7.3 µg/mL) and mouse 17B4 antibody was tested at 263 nM (39.5 µg/mL). Primary antibody was pre-incubated with varying concentrations of peptide, for at least 60 min at 4 °C with mixing, before being incubated on-slide for 16 min at 37 °C. Each peptide was tested, in duplicate, at concentrations of 5000, 500, and 50 nM. Slides were scored by a qualified reader using the intensity of the DAB stain using a common 0 (no signal) to 3 (most intense) scale, and the percent of tumor tissue that is stained. LAG3 positive T cells typically display a punctate cytoplasmic staining pattern. Macrophages and plasmacytoid dendritic cells can also be stained with a lighter diffuse cytoplasmic pattern, however, for the purposes of this assay, that cell population is not included in the analysis. Digital images were captured on an Aperio imager at 40× magnification.

Breast cancer specimens, including normal areas which were the margin tissue, were obtained from patients at the time of surgery. The archival specimens were obtained from the Zhongshan Hospital of Traditional Chinese Medicine Affiliated to Guangzhou University of Chinese Medicine. The sections (4-µm) were mounted onto silane-coated slides. Immunohistochemistry was automatically performed using the Ventana XT System Discovery® (Roche). Briefly, tissue sections were treated with protease I (Roche) at 37̊C for 16 min for antigen retrieval. The following primary antibody was used: KRT81 polyclonal antibody was diluted at 1:25 (Abcam), and sections were incubated at 37̊C for 32 min. The tissue sections were then incubated at 37̊C for 20 min with a universal secondary antibody (Affinity). The site of peroxidase binding was determined using the DISCOVERY DABMap Detection kit (Roche). Sections were then counterstained with Hematoxylin II (Roche) for microscopic examination. As a negative control, nonimmune γ-globulin was used instead of the antibody. The specimens were observed and photographed using a fluorescence microscope FSX100 (Olympus).

Hamster lungs were harvested in 4% PFA and stored at 4 °C. The tissue was dehydrated, fixed, and infiltrated with paraffin on the Leica Polaris 2 tissue processor. The infiltrated tissue was then embedded and sectioned at 5 microns. Antigen retrieval was performed on the Roche Ventana Discovery ULTRA staining platform using Discovery CC1 (Roche, Indianapolis, IN, USA; Cat#950-500) for a total application time of 64 min. The primary antibody, human Anti-SARS-CoV-2 Nucleocapsid monoclonal (E16C) (Thermo Fisher, Corning, NY, USA; MA1-7403, 0.1 mg/mL), was incubated at a 1:100 dilution, at room temperature, for 45 min. Secondary immunostaining used a horseradish peroxidase (HRP) multimer cocktail (Roche, Indianapolis, IN, USA; Cat#760-500), and immune complexes were visualized using the ultraView Universal DAB (diaminobenzidine tetrahydrochloride) Detection Kit (Roche, Indianapolis, IN, USA; Cat#760-500). Slides were then washed with a Tris-based reaction buffer (Roche, Indianapolis, IN, USA; Cat#950-300) and counter-stained with Hematoxylin II (Roche, Indianapolis, IN, USA; Cat #790-2208) for 4 min. Scoring was performed as shown in Table S2.

Nestin protein levels was detected using the ultraView DAB protocol on the automated VENTANA® BenchMark ULTRA platform (Roche, Basel, Switzerland).
To detect nestin expression slides were incubated with 1:200 diluted anti-nestin antibody, clone 10C2 (#MAB5326, RRID:AB_11211837, MerckMillipore, Burlington, Massachusetts, USA), for 32 min. VENTANA® standard signal amplification and ultra-wash was followed by counterstaining with Hematoxylin II (#790-2208, Roche, Basel, Switzerland) and blueing reagent (#760-2037, Roche, Basel, Switzerland) for 4 min each. Slides were removed from the staining platform, washed with tap water containing a drop of dishwashing detergent and rinsed with deionized water. After staining, all specimens were immersed in a series of ethanol (EtOH) solutions (#20821.330, VWR, part of Aventor, Radnor, Pennsylvania, USA) of increasing concentrations until 100% and Xylol (#534056-4L, Sigma, part of Merck, Darmstadt, Germany). Eukitt® (#6.00.01.0001.06.01.01, ORSAtec GmbH, Bobingen, Germany) was used for mounting.

To detect COL11A1 expression, IHC was performed on Ventana Discovery Ultra autostainer (Roche, Indianapolis, IN) with the following protocol and reagents. Vial of mAb anticol11A1 (Oncomatryx, High Concentration 2.3 mg/mL Rabbit monoclonal (Clone 1e8.33)). All reagents were provided by Roche, Indianapolis IN: Samples underwent heat-induced epitope retrieval, (Cell Conditioning Solution ULTRA CC1 (950-224, Roche)) for 32 min; the primary antibody was incubated for 30 min per manufacturers instruction; followed by DISCOVERY OmniMap anti-Rt HRP (760-4457; Roche), DISCOVERY ChromoMap DAB kit (760-159; Roche), Hematoxylin II (790-2208; Roche), and Bluing reagent (790-2037; Roche) were used for visualization. All samples (PDX, xenograft, primary, normal) were stained in along with positive control (LM7 xenograft) and negative control xenograft (143B COL11A1 KO xenograft), grown subcutaneously in NSG female mice, and tumors were harvested when they reached a size of 1000 mm³. Isotype controls were used as well.