Dako target retrieval solution

Tissues were fixed in 10% formalin, embedded in paraffin, and sectioned at 4 µm intervals. For immunofluorescence, slides were heated in a pressure cooker using DAKO Target Retrieval Solution (Agilent Technologies, cat. no. S170084-2), blocked for 1 h at RT with Innovex Background Buster (Innovex, cat. no. NB306) with 5% Fc Receptor Block (Innovex, cat. no. NB309) and incubated with primary antibodies against CD3 (Santa Cruz Biotech, cat. no. sc-20047), CD8α (Santa Cruz Biotech, cat. no. sc-7188), or Granzyme B (abcam, cat. no. ab134933) at 1:100–200 overnight at 4°C. Slides were mounted in a DAPI containing medium (Santa Cruz) and visualized using either Alexa Fluor 488 (abcam, cat. no. ab150113) or Alexa Fluor 594 (abcam, cat. no. ab150080) conjugated secondary antibodies.

Tissues were fixed in Mildform 10N (FUJIFILM Wako) overnight at 4 °C and paraffinized. Sections of 3 µm thickness were assessed after HE staining under a light microscope. For immunofluorescence analysis, sections were deparaffinized and incubated with Dako target retrieval solution (Agilent Technologies, Santa Clara, CA, USA) buffer at 110 °C for 10 min in an autoclave. After blocking with Carbo-Free Blocking Solution for 1 h, sections were incubated overnight at 4 °C with primary antibodies or lectin. Alexa Fluor conjugated secondary antibodies (Invitrogen, Waltham, MA, USA) were used for immunodetection. The details regarding the antibodies and lectins used are described in Table S1. Nuclei were counterstained with Hoechst 33,342 (Thermo Fisher Scientific, Waltham, MA, USA). Images were captured using a BIOREVO BZ-X800 microscope system (Keyence, Higashiyodogawa, Osaka, Japan).

The IF analysis was performed on paraffin sections (thickness 3 μm) using a conjugated anti-CD248 antibody (Novus Biologicals, Littleton, CO, USA). Antigen retrieval was carried out using Dako Target Retrieval solution (Agilent Technologies, Santa Clara, CA, USA). Vasculature pericytes were highlighted using a Cy3-conjugated anti-α-SMA antibody (Sigma-Aldrich, St. Louis, MO, USA) and EC using unconjugated anti–von Willebrand factor (vWF) antibody (Dako, Glostrup, Denmark). The immunoreaction was revealed using secondary antibody (Alexa Fluor; Life Technologies, Carlsbad, CA, USA). Cell nuclei were visualized using 4′,6-diamidino-2-phenylindole. Fluorescence was analyzed using a BX53 fluorescence microscope (Olympus, Center Valley, PA, USA). The intensity of fluorescence was measured using ImageJ software (National Institutes of Health, Bethesda, MD, USA).

Sections 4 µm thick were obtained from the most representative formalin-fixed, paraffin-embedded (FFPE) blocks of each NSCLC tumor for analysis. Immunohistochemical staining was performed using the standard technique of antigen retrieval and development of avidin–biotin–peroxidase complexes (ABC). Briefly, 4-µm tissue sections were deparaffinized in xylene and mounted on Poly-L-lysine-coated slides. All slides were subjected to a heat-based antigen retrieval method using DAKO Target Retrieval Solution (Agilent, Santa Clara, CA, USA), containing 10 mM citrate buffer (pH 6), and a water bath (95–99 °C) for approximately 20 min before immunostaining. The primary antibody to TTF-1, DAKO clone 8G7G3/1 (Agilent, Santa Clara, CA, USA), was used at the manufacturer’s recommended dilution (1:200). Slides were counterstained with hematoxylin. Sections of TTF-1-positive LUAD were used as positive controls. The primary antibody was replaced with diaminobenzidine (3,3′-diaminobenzidine) solution for the negative controls. TTF-1 nuclear staining was graded as negative (<5%), weak positive + (5–49%), or strong positive ++ (>50%) based on the percentage of tumor nuclei with unequivocal staining.

For PD-1 immunofluorescence staining, paraffin sections were deparaffinized in xylene, rehydrated with a decreasing series of ethanol concentrations and then washed with deionized water. The slides were then immersed in citrate buffer (Dako Target Retrieval Solution, Agilent, Cat# S1699) and boiled for 20 min, followed by 30 min of permeabilization with 0.25% Triton in PBS at room temperature. The sections were incubated with a blocking solution containing 5% goat serum in PBS for one hour at room temperature before the anti-rabbit PD-1 antibody (Abcam, Cat# ab214421, 1:150) was added and incubated overnight at 4 °C. The slides were then washed with 0.025% PBS-Tween and stained with goat anti-rabbit Alexa Fluor-568 secondary antibody (Thermo Fisher Scientific, Cat# A11034, 1:200) for one hour at room temperature. The nuclei were counterstained with DAPI for 15 min, washed in PBS, and then mounted with Fluoromount aqueous mounting medium (Sigma-Aldrich, Cat# F4680). Images were captured with a Hamamatsu Nanozoomer scanner, and the fluorescence intensity in regions of healthy and tumor tissues in the cross-sections was quantified by ImageJ. After splitting the different color channels, PD-1 fluorescence intensity (magenta color channel) was quantified in tumor areas and healthy areas in each section, and the placebo- and BAMLET-treated groups were compared.