Anti panck

The cryosections of lung biopsy samples from NSCLC patients were prepared by standard OCT-based method. The sections from tumor-rich or tumor-poor areas of the same patient were processed for immunofluorescence staining using anti-CD3 (Biolegend), anti-CD8 (Abcam), anti-panCK (Abcam), and anti–TLT-1 (R&D Systems) primary antibodies followed by immunofluorophore-tagged secondary antibodies (Alexa Fluor 488 for CD3 primary, Alexa Fluor 594 for TLT-1 primary, BV-421 for CD8 primary, and Cy-5 for panCK primary). The stained sections were viewed under a spinning disk confocal microscope (Nikon Eclipse Ti) under the same software settings for all sections to be compared and images were captured using Volocity v6.3.
The dual immunohistochemistry (IHC) was performed by using kit-based method for dual staining as per the manufacturer’s instructions (Vector Biosciences). The two primary antibody combinations i.e., CD4 and TLT-1 or CD8 and TLT-1 were used followed by conjugated secondary antibodies and counterstaining using Gill’s hematoxylin stain. The tumor sections were observed under a Nikon 80i microscope. The tumor nests were used for quantification by the observer blinded to experimental groups in consultation with a pathologist.

Paraffin sections of colon cancer tissues were collected and used for mIHC with an Opal 7-colour kit (PerkinElmer, NEL811001KT). For the first and subsequent rounds antigen epitope retrieval was performed in EDTA (pH 8.0) buffer using a microwave at the lowest power for 15 min. The slides were cooled to room temperature, and washed with TBST (three times, 3 min). Then, the slides were blocked with antibody diluent/block buffer for 10 min and incubated with primary antibody at 4 °C overnight. The following primary antibodies were used: anti-pan-CK (Abcam, Cambridge, UK, ab27988,); anti-HDAC6 (Cell Signaling Technology, USA, 7558); anti-CD68(Invitrogen, CA, USA, 14-0688-82); and anti-CD163(Abcam, ab182422). The next day, the slides were placed at room temperature and incubated with HRP-conjugated secondary antibody for 10 min after being washed with TBST three times. The slides were treated with TSA dye for 10 min, and then microwaved for epitope retrieval to label with the next antibody. Finally, the nuclei were stained with DAPI for 10 min at room temperature.

Antibodies such as anti-CD8 (CST70306, Cell Signaling Technology, United States), anti-CD56 (CST3576), anti-panCK (CST4545), anti-S100 (ab52642, Abcam, UK), anti-HLA-DR (ab92511), and anti-CD68 (BX50031, Biolynx, China) were used in mIF using the PANO 7-plex IHC kit (Panovue, Beijing, China), according to the manufacturer’s instructions. The Mantra System (PerkinElmer, Waltham, MA, United States) was used to scan the stained slides and reconstruct the images of the sections. inForm image analysis software (PerkinElmer, Waltham, MA, United States) was used to quantify the cells in the images.

Primary antibodies used in this study are anti-Top1 (Abcam, ab109374), anti-BRD4 (Thermo Fisher Scientific, A301985A), anti-H3K36me3 (Abcam, ab9050), anti-H3K27ac (Abcam, ab177178), anti-CSTF64 (Thermo Fisher Scientific, A301092A), anti–RNAPII–Ser2-P (Abcam, ab5095), anti–total RNAPII (Abcam, ab817), anti-γH2AX for in vitro IF (Sigma-Aldrich, 05-636), anti-γH2AX for mIF (Cell Signaling Technology, 9718S), anti-p27 (Abcam, ab32034), anti-BrdU (Becton Dickinson, 555627), anti–cleaved caspase 3 (Cell Signaling Technology, 9664 L), and anti-PanCK (Abcam, ab6401). Secondary antibodies are anti-mouse (Thermo Fisher Scientific, A11029) and anti-rabbit (Thermo Fisher Scientific, A32790) Alexa Fluor 488–conjugated antibodies.

Primary antibodies used in this study are anti-Top1 (Abcam, ab109374), anti-BRD4 (Thermo Fisher Scientific, A301985A), anti-H3K36me3 (Abcam, ab9050), anti-H3K27ac (Abcam, ab177178), anti-CSTF64 (Thermo Fisher Scientific, A301092A), anti–RNAPII–Ser2-P (Abcam, ab5095), anti–total RNAPII (Abcam, ab817), anti-γH2AX for in vitro IF (Sigma-Aldrich, 05-636), anti-γH2AX for mIF (Cell Signaling Technology, 9718S), anti-p27 (Abcam, ab32034), anti-BrdU (Becton Dickinson, 555627), anti–cleaved caspase 3 (Cell Signaling Technology, 9664 L), and anti-PanCK (Abcam, ab6401). Secondary antibodies are anti-mouse (Thermo Fisher Scientific, A11029) and anti-rabbit (Thermo Fisher Scientific, A32790) Alexa Fluor 488–conjugated antibodies.