Bond epitope retrieval solution 1

SK6005 skin tumor tissues were fixed and embedded in paraffin, and 4 µm sections were prepared for staining. Immunohistochemical staining was performed with primary antibodies specific for CD4 (Sino Bio 50134-R001), CD8 (Affymetrix 14–0808), and FoxP3 (Novus NB100–39002) on the Bond RX system (Leica Biosystems, Germany). Briefly, the sections were processed by the following incubation steps: Bond dewax solution (Leica AR9222), 0.5 min at 72 °C; Bond epitope retrieval solution 1 (Leica AR9961) or Bond epitope retrieval solution 2 (Leica AR9640), 20 minutes at 100 °C; Bond wash buffer (Leica AR9590), 3 minutes at room temperature (RT); peroxide block (Leica DS9800), 10 minutes at RT; goat serum, 20 minutes at RT for the anti-CD8 antibody; primary antibody, 60 minutes at RT; Bond wash buffer, 3 times for 2 minutes each at RT; polymer, 20–30 minutes at RT; Bond wash buffer, 3 times for 2 minutes each at RT; DAB (Leica DS9800), 5 minutes at RT; and hematoxylin (Leica DS9800), 10 minutes at RT. Five fields in each stained sample without necrosis were randomly selected and imaged at 20× magnification. All images were analyzed with ImageJ software. Positive cells were counted, and the average number of positive cells in 5 fields was taken as the score value of each sample.

A Leica Bondmax Stainer in modified protocol F was used to stain for Slfn12 protein in the gastric bypass and EGD tissue sections. Paraffin sections were dewaxed and antigen retrieval was performed with Leica Bond Epitope Retrieval Solution 1 for 20 min. For immunostaining, slides were sequentially washed with bond wash buffer (BWB), incubated with SLFN12 antibody (Rabbit polyclonal, LS-B4757, 1:200 dilution; LSBiosystems, Seattle, WA, USA) for 30 min, bonded after the primary antibody for 20 min, washed with BWB, bonded with polymer for 20 min, washed with BWB, bonded with peroxidase block for 10 min, washed with BWB, bonded with DAB for 10 min, washed with deionized water, bonded with hematoxylin for 5 min, and washed with BWB. The slide was dehydrated, and a coverslip was applied. Slides were then deidentified as to the peri-operative or post-operative nature of the specimens. Two blinded observers were asked to score the intensity of epithelial SLFN12 staining on a 0 to 3 scale in each image based upon a predetermined scale. These scores were then aggregated and subjected to statistical analysis by a Student t-test in a GraphPad Prism 8.

Upon collection, intestinal tissues were immediately placed in 10% buffered formalin. After processing, the paraffin-embedded sections were mounted on slides. Sections were dewaxed with Leica Bond Dewax Solution and antigen retrieval performed using Bond Epitope Retrieval Solution 1 (Leica) for 20 min at 100 °C. Slides were incubated with 3% hydrogen peroxide for 5 min at room temperature and visualized by using an automated procedure on a NexES IHC Staining Module (Ventana Medical). A rabbit anti-CD13 (ANPEP, APN) polyclonal antibody (Abcam) prepared against a peptide covering amino acids 400–500 of human CD13 was used for the detection of ANPEP antigen. The antibody was diluted 1:3200 in Bond Primary Antibody Diluent (Leica) and incubated on slides for 15 min at room temperature. Slides were washed and bound antibody detected with anti-Rabbit IgG HRP, which was included in the kit. HRP activity was visualized with DAB and slides counter stained with hematoxylin. The PEDV and TGEV IHC were performed by using similar methods as a routine diagnostic test by the Kansas State University and University of Missouri veterinary diagnostic laboratories.

Formalin fixed, paraffin embedded tissue sections were deparaffinized using Bond Dewax Solution (Leica) after which Epitopes were retrieved using Bond Epitope Retrieval Solution 1 (Leica). The sections were stained for EV-A71 antigen (10F0, abcam), PSGL1 (bs-0561R, Bioss) or SCARB2 (NB400-129, Novus). In addition, haematoxylin and eosin staining was performed to visualize inflammation.

FFPE sections of cohort PCa2 were stained on a BOND MAX autostainer (Leica). Briefly, paraffin-embedded sections were first dewaxed, and antigen retrieval was performed in BOND epitope-retrieval solution 1 (Leica). Mouse monoclonal 3F2 anti-GSTP1 (1:2000, 3369) and rabbit monoclonal anti-ERG (1:100, ab92513) were purchased from Cell Signaling Technology (Danvers, MA) and Abcam (Cambridge, UK), respectively. Slides were analyzed by light microscopy, reviewed and scored by an uropathologist, according the Allred method [17 (link)]. The Allred score is a semi-quantitative system that takes into account the proportion of positive cells (scale of 0–5) and staining intensity (scale of 0–3). The proportion and intensity score were summed to obtain the total scores of 0, 2–8. A score of 0–2 was considered as negative, whereas 3–8 was taken as positive [17 (link)].

Immunohistochemistry was performed on formalin-fixed, paraffin-embedded sections (4 μm) using an automated immunostainer with cover tile technology (Bond-III system, Leica Biosystems). The commercial antibody anti-ANO4 (Sigma, HPA053412) and anti-CYP11B2 antibody (a kind gift from Dr Celso E. Gomez-Sanchez) were used as the primary antibodies. The antigen retrieval was carried out using the combination of heat and Bond Epitope Retrieval Solution 1 (Leica, AR9961) for 20 minutes. The optimal working dilution for ANO4 was 1/50. The Bond Polymer Refine Detection kit (Leica, DS9800) was used for detecting and visualizing the antigens. Negative controls, in which primary antibodies were omitted, resulted in a complete absence of staining.