Dako retrieval buffer

Tissues were fixed in 10% formalin, paraffin-embedded, and sections at 4μm interval were cut from each tissue, and stained with hematoxylin and eosin (H&E), or via immunohistochemistry (IHC) or immunofluorescence (IF). For immunohistochemistry, slides were deparaffinized by xylenes and rehydrated by ethanol gradient, then heated in a pressure cooker using DAKO retrieval buffer (DAKO, Santa Clara, CA). Endogenous peroxidases were quenched in 3% hydrogen peroxide in methanol for 30 minutes. Tissues were blocked with 0.5% BSA in PBS for 30 minutes and incubated with primary antibodies against: SMAD4, CD3 (Santa Cruz), CD45, or PD-L1 (Cell Signaling) at 1:50–1:200 overnight at 4°C. Slides were developed using HRP-conjugated secondary antibodies followed by DAB substrate/buffer (DAKO).
For immunofluorescence, slides were heated via pressure cooker in DAKO retrieval buffer and tissues blocked with 0.5% BSA in PBS for 1 hour at room temperature. Sections were exposed to primary antibodies against CK19 (University of Iowa Hybridoma Bank), E-Cadherin (Cell Signaling), CD3, (Santa Cruz), or IFNγ (abcam) at 1:50–1:200 overnight at 4°C. Slides were developed using AlexaFluor 488- or 594-conjugated secondary antibodies (1:200–1:1,000, abcam), mounted in DAPI-containing media (Santa Cruz Biotechnology), exposed to DAPI, FITC, and Texas Red filters.

Tissues were fixed in 10% formalin, paraffin-embedded, and sections at 4 mm interval were cut from each tissue, and stained with hematoxylin and eosin (H&E) or via immunohistochemistry (IHC). For immunohistochemistry, slides were deparaffinized by xylenes and rehydrated by ethanol gradient, then heated in a pressure cooker using DAKO retrieval buffer (DAKO, Santa Clara, CA). Endogenous peroxidases were quenched in 3% hydrogen peroxide in methanol for 30 min. Tissues were blocked with 0.5% BSA in PBS for 30 min and incubated with primary antibodies against: P16, CD45, CD3, CD68, or Neutrophil Elastase (abcam, Cambridge, MA) 1:100–1:200 overnight at 4 °C. Slides were developed using HRP-conjugated secondary antibodies followed by DAB substrate/buffer (DAKO, Santa Clara, CA).

For immunohistochemical study, OSCC tissues were deparaffnized using xylene and then rehydrated through an ethanol series. Antigens were retrieved by autoclaving the slides in Dako retrieval buffer (Dako, Carpinteria, CA, USA). After cooling to room temperature, the slides were incubated with primary ADHFE1 antibody (Sigma-Aldrich, St. Louis, MO, USA) or ALDH1A2 antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA) at 4 °C overnight. Specific signals were then developed with LSAB+ kit (Dako) using diaminobenzidine as chromogen. Sections were then counterstained with hematoxylin and observed under light microscope. Tumor ADHFE1 and ALDH1A2 level were scored according to staining intensity as follows: 0, negative; 1, weak; 2, intermediate; and 3, strong. Two pathologists independently assessed all the scorings.