Mantra system

The obtain multispectral images, the stained slides were scanned using the Mantra system (Perkin Elmer), which captures the fluorescent spectra at 20-nm wavelength intervals from 420 to 720 nm with identical exposure time. The scans were combined to build a single stack image. Images of unstained and single-stained sections were used to extract the spectrum of autofluorescence of tissues and each fluorescein, respectively. The extracted images were further used to establish a spectral library required for multispectral unmixing by InForm image analysis software (PerkinElmer). Using this spectral library, we obtained reconstructed images of sections with the autofluorescence removed.

Six pairs of lymph nodes were randomly selected, formalin-fixed, paraffin-embedded, and subjected to multiplex immunohistochemistry (mIHC). The mIHC was performed by using a PANO 7-plex IHC kit (Panovue, China) following the standard protocol [16 (link)]. Immune cell panels included the following antibodies: CD3 (1 : 200, Abcam, ab16669), CD8A (1 : 300, Cell Signaling Technology, 70306), Foxp3 (1 : 500, Abcam, 20034), PD1 (1 : 50, Cell Signaling Technology, 43248), and CD163 (1 : 100, Cell Signaling Technology, 93498). The slides were incubated with the primary antibodies, followed by 0.3% hydrogen peroxide solution for blocking endogenous peroxidase. DAPI (Sigma-Aldrich) was used for nuclear counterstaining. Images were acquired and analyzed by using a Mantra System (PerkinElmer) and inForm image analysis software (PerkinElmer), respectively.

Consecutive sections from three human cervical squamous cell carcinoma tissue arrays containing 93 intact cervical carcinoma tissues and paired normal adjacent cervical tissues were purchased from Shanghai Outdo Biotech Co., Ltd. (OD-CT-RpUtr03-004, OD-CT-RpUtr03-005, and OD-CT-RpUtr03-006). The sections were stained with anti-GABRA2 antibody (Thermo-Fisher, #PA5-26305) at a 1:100 dilution, anti-ZNF257 antibody (Thermo-Fisher, #PA5-36012) at a 1:100 dilution, anti-SLC5A8 antibody (Proteintech, #21433-1-AP) at a 1:100 dilution, and anti-RAB3C antibody (Proteintech, #10788-1-AP) at a 1:200 dilution. After washing, the sections were incubated with biotin-conjugated secondary antibodies and subsequently with streptavidin–HRP. The sections were finally visualized by incubation with 3,3′-diaminobenzidine (DAB) substrate. Images were obtained with the Mantra System (PerkinElmer, Massachusetts, USA) with identical exposure times. The H-score was used for quantifying the protein levels of GABRA2, ZNF257, SLC5A8, and RAB3C in normal and tumor tissues, and this score was calculated by multiplying the staining area (scored as the percentage of differentially stained area) and the staining intensity (weak, moderate, and strong were scored as 1, 2, and 3 based on color density). Student’s t test was performed for the statistical analysis.

The 4 mm thick FFPE tissues were deparaffinized in xylene before they were rehydrated in gradient concentrations of ethanol. In antigen retrieval step, the samples were incubated in sodium citrate buffer (PH = 6.0) for 4 min. Hydrogen peroxide was used to block the endogenous peroxidase activity for 10 min. The samples were covered with the primary antibodies (ASCL1, Abcam, ab213151; NeuorD1, Abcam, ab60704; YAP1, CST, 14074; POU2F3, Abcam, ab191840; CD44, CST, 3570). After incubation with the secondary antibodies for IHC, the samples were dehydrated, covered with coverslips as previously done.^[49] For multiplex immunohistochemistry, PANO 4‐plex IHC kit (Panovue, Beijing, China) was used. Panel 1 antibodies (CD3, Abcam, ab17143; CD8, CST, 70306; HAVCR2, CST, 45208), and panel 2 antibodies (CD16, Abcam, ab246222; CD56, CST, 99746S; TGFBR2, Abcam, ab78419) were applied sequentially. And Mantra System (PerkinElmer, Waltham, Massachusetts, US) was utilized to obtain multispectral images.

IHC was performed using an anti-CHAF1A antibody (ab126625), with a 2-step protocol. Specialized pathologists calculated the number of positively stained cells and the staining intensity to create grade categories under a microscope. A previously reported semi-quantitative method was used to assess IHC scores [22 (link)]. mIF staining was conducted using the PANO 7-plex IHC kit (Panovue, Beijing, China), section images were reconstructed using the Mantra System (PerkinElmer, Waltham, MA, USA), and quantification of cells in the images was performed using the inForm image software (PerkinElmer). Anti-CD8 (CST70306; Cell Signaling Technology, USA), anti-CD56 (CST3576), anti-CD68 (BX50031; Biolynx, China), anti-HLA-DR (ab92511), anti-panCK (CST4545), and anti-S100 (ab52642) antibodies were used for staining.