Ab52632

Nine prostate cancer tissue microarrays consisting of 97 patients (n = 1547 cores) were obtained from the Pathology Department at the University of California, Irvine as previously reported [22 (link)].
Blocks from each TMA were stained with three antibodies—CD11c, OX40L, and CTLA4 by CrownBio (Per CrownBio Inc., San Diego, CA, USA). Immunohistochemistry was performed on a Bond RX autostainer (Leica Biosystems) with heat induced epitope retrieval in an EDTA buffer (pH 9.0) using the standard protocol. The primary antibodies used were rabbit monoclonal CD11c antibody (Abcam, ab52632, 1:100), rabbit monoclonal OX40L antibody (Cell Signaling Technology, 59036, 1:100, Danvers, MA, USA), and rabbit monoclonal CTLA4 antibody (Abcam, ab237712, 1:100, Cambridge, UK). Bond polymer refine detection (Leica Biosystems, DS9800, Wetzlar, Germany) was used as a secondary antibody detection system according to the manufacturer’s standard protocol. After staining, sections were dehydrated and film coverslipped using a TissueTek-Prisma and Coverslipper (Sakura). Whole slide scanning (40×) was performed on a NanoZoomer Digital Slide System NDP2.0-HT (Hamamatsu, Shizuoka, Japan).

Paraffin-embedded, formalin-fixed LMS and LPS specimens were used for IHC. Sections were incubated overnight in a humidified container at 4°C with the primary antibodies of MUC1 (1:100, ab109185; Abcam) and CD11c (1:100, ab52632, Abcam). After three times washing, tissue sections were incubated with the secondary antibody conjugated with streptavidin–horseradish peroxidases for 1 h at room temperature. The slides were stained with 3, 3-diaminobenzidine tetrahydrochloride (DAB) and the nuclei were counterstained with hematoxylin. Immunostaining on each slide was assessed by experienced pathologists to examine the percentage of MUC1 or CD11c positive tumor cells and presented as histochemistry score (H-score). H-score = Σpi(i+1) where i is the intensity score and pi is the percent of the cells with that intensity.

IHC analysis was implemented to assess the DRD5 (ab32620, Abcam, Cambridge, U.K.), SLC2A14 (QC354Hu01, QchengBio, Shanghai, China), IGF1 (ab263903, Abcam), CD11c (ab52632, Abcam), and PD-L1 (ab205921, Abcam) expression levels following manufacturers’ protocols and previously described procedures (34 (link), 35 (link)). The BCa samples were scored according to the degree of cell staining intensity and density.

Tissue microarrays (TMAs) were constructed with FFPE blocks of archived tumor specimens of the recruited 98 ESCC patients. Two 1-mm cores from representative areas of each tumor sample were punched and arrayed onto a recipient paraffin block. Tissue sections (4 µm thick) obtained from TMA blocks were subjected to multiplex fluorescent immunohistochemistry (mfIHC) staining using the PANO Multiplex IHC kit(0004100100, Panovue, Beijing, China) to examine specific cell markers including CD11c (ab52632, Abcam, Cambridge, UK), CD45RO (55618, Cell Signaling, Danvers, MA, USA), CD68 (ZM0060, ZSGB-Bio), panCK (4545, Cell Signaling), and PD-L1 (13684, Cell Signaling) in panel A and CD4 (BX50023, BioLynx, Brockville, ON, Canada), CD8A (70306, Cell Signaling), CD56 (3576, Cell Signaling), FoxP3 (320202, BioLegend, San Diego, CA, USA), and granzyme B (ab4059, Abcam) in panel B. Different primary antibodies were sequentially applied, followed by horseradish peroxidase-conjugated secondary antibody incubation and Tyramide signal amplification (TSA). After each TSA operation, the slides were microwave heat-treated. Nuclei were stained with 4′,6′-diamidino-2-phenylindole (DAPI, D9524, Sigma-Aldrich, St. Louis, MO, USA) after all the human antigens have been labeled (Pan et al., 2021 (link)).

The sections were stained using the Ventana BenchMark Ultra Slide Staining system (Roche, Switzerland), with the use of the UltraView Universal DAB and UltraView Universal AP RED Detection kits (Roche, Switzerland). The procedure consisted of heating at 72°C for 8 min., then at 97°C under a high pH for 64 min. The incubation with the mouse anti-human monoclonal anti-MK (sc-46701, ID AB_627949) antibody (Santa Cruz, USA) at dilutions 1/25, 1/50 or 1/100 for 2 hrs. was followed by a 40 min. incubation with rabbit anti-human monoclonal anti-CD11c (ab52632, ID AB_2129793), anti-CLEC4C (ab239077, ID AB_2904588) or anti-CD68 (ab213363, ID AB_2801637) antibodies (Abcam, UK). The dilutions of these antibodies were according to the manufacturer’s instructions. Counterstaining with hematoxylin was performed during 16 min. Positive cells were observed in at least five high power fields (diameter of the field = 0.44 mm) with 60x objective and 10x ocular lenses (Olympus BX53). Positive cell identification was done by four independent expert/trained readers.

Tissue sections of 15 μm were mounted on glass slides (Superfrost Plus Green). Slides were placed for 45 min into an oven (70°C) to remove excess paraffin. Tissues were rehydrated with a Xylol/ethanol train of solvents. Antigen retrieval was performed using citrate buffer pH 6.0 (sodium citrate 10 μM) at 90°C for 20 min. Then, samples were permeabilized with a solution of 10 mg/mL bovine serum albumin, 5% horse serum, 0.02% sodium azide, and 0.3% Triton for 2 h. Following permeabilization, samples were incubated with different primary antibodies: rabbit anti-human CD11c (ab52632, Abcam), rat anti-human HLA-DR (YD1/63.4.10, Invitrogen), mouse anti-human BDCA-3 (ab6980, Abcam), rat anti-human CD40 (ab22469, Abcam), and rabbit anti-human IDO (ab122402, Abcam). Primary antibodies were revealed with secondary conjugated antibodies: anti-rabbit Alexa Fluor 488 (711-547-003, Jackson ImmunoResearch), anti-rat Alexa Fluor 594 (712-585-153, Jackson ImmunoResearch), and anti-mouse Alexa Fluor 647 (715-605-151, Jackson ImmunoResearch). After that, nuclei were stained with Hoechst (Invitrogen) for 10 min. Sections were mounted with 10% glycerol in PBS. Images were acquired after this step of staining and used for analysis. To perform the second round of staining, coverslips were removed by soaking the slides in 1X PBS, and samples were processed as described below.