Ab78237

IHC staining was carried out on 4 μm-sections of formalin-fixed paraffin-embedded (FFPE) tissue obtained from patients with pCCA. Xylene was used to deparaffinize the sections, which were then subjected to hydration using an ethanol series. Epitope retrieval was accomplished by boiling the sections in citrate buffer (pH 6.0) or Tris-EDTA (pH 9.0) under high pressure for 10 min and then leaving them to self-cool. Endogenous peroxidase was quenched for 10 min using 3% hydrogen peroxide, followed by blocking of nonspecific binding by incubation for 30 min in 5% bovine serum albumin (BSA). The sections were then incubated with primary antibodies: rabbit anti-CD20 (ab78237, Abcam, Cambridge, UK), rabbit anti-CD21 (ab75985, Abcam, Cambridge, UK) overnight at 4 °C. After washing, the sections were incubated with biotin-labeled secondary antibodies (K5007, Dako at Agilent, Santa Clara, CA, USA) at 37 °C for 30 min. Finally, the sections were washed with phosphate-buffered saline (PBS) and stained for 3–10 min using diaminobenzidine (DAB) solution according to which primary antibody was used, followed by counterstaining using hematoxylin. The whole slides were scanned using a Vectra^® Polaris™ instrument (Akoya Biosciences, Menlo Park, CA, USA).

FFPE tissue sections were stained with the PANO 7‐plex IHC kit (Panovue) according to the manufacturer's instructions, including deparaffinisation with xylene, hydration with gradient ethanol, antigen retrieval with sodium citrate buffer (0.01 M, pH = 6.0), removal of endogenous peroxidase with H₂O₂, blocking tissues with 10% goat serum and staining with antibodies and TSA‐RM. The next cycle of staining started with antigen retrieval and was stopped with TSA labelling. The antibodies used included CD4 (ab133616, 1:500; Abcam) with Opal 620, CD8 (C8/144B, 1:200; CST) with Opal 690, and CD20 (ab78237, 1:2000; Abcam) with Opal 520. The antibodies used included CD45 (20103‐1‐AP, 1:2000; Proteintech) with Opal 570, SFRP4 (15328‐1‐AP, 1:200; Proteintech) with Opal 620, and IgG (EPR4421, 1:500; Abcam) with Opal 520. Then, all slides were scanned and analysed using the Vectra Automated Quantitative Pathology Imaging System (Vectra Polaris featuring MOTiF™). Finally, the percentage of SFRP4⁺ AFCs around the Tumour area was analysed in the HALO software (version 3.3). Specifically, the typical tumour area were identified based on DAPI staining, the distance and the area of 0–2, 2–4 and >4 mm from the tumour area were delineated, and the percentage of SFRP4⁺ AFCs in DAPI⁺ cells were calculated and statistical analysed.

First, we deparaffinized the sections in xylene and then hydrated by graded series of ethanol, the detail of the staining procedure were mentioned in our previous study^{19 (link)}. Following primary antibodies were used to incubate the sections for overnight at 4 °C. Primary antibodies included rabbit anti-CD3 (Clone SP7; ab16669, Abcam, Tokyo, Japan, 1:200 dilution), rabbit anti-CD20 (Clone EP459Y; ab78237, Abcam, Tokyo, Japan, 1:100 dilution), mouse anti-CD163 (Clone 10D6; Novocastra, Newcastle, UK, 1:400 dilution), mouse anti-CD204 (Clone SRA-C6; TransGenic Inc, Kumamoto, Japan, 1:200 dilution), mouse anti-CD206 (Clone 5C11; Abnova, Taipei, Taiwan, 1:300 dilution), and rabbit anti-EGF (Clone ab9695; Abcam, Tokyo, Japan, 1:30 dilution). Antibody was washed by TBST and as a chromogen 100–400 µl DAB (Peroxidase Stain DAB Kit, nacalai tesque, Kyoto, Japan) was applied to each section. Finally, Mayer’s hemalum solution (Merck KGaA, Darmstadt, Germany, 1:4 dilution) was used for counterstain and then sections were washed two times for 5 min each in dH₂O. After dehydration, sections were mounted with coverslips. The numbers of CD3-, CD20-, CD163-, CD204-, CD206-, and EGF-positive cells were counted in 4 mm² sections from five independent high-power microscopic fields (400×, 0.0625 μm²).

Paraffin-embedded section (3 µm thick) were dewaxed in xylene and heated in 0.01 M sodium citrate buffer (pH = 6.0) in a microwave at 60 °C for 20 min for antigen retrieval. Tissue sections were then blocked in 0.3% H₂O₂ for 10 min to remove endogenous peroxidase activity, followed by incubation with primary antibody against CD4 (1:1000; ab288724, abcam, Cambridge, MA, USA), CD20 (1:50; ab78237, abcam), or CD138 (1:100; ab130405, abcam). The target was visualized with a DAB staining kit (GK600710, Gene Tech, Shanghai, China).