Pathological section scanner

The liver sections were deparaffinized and rehydrated for immunohistochemical staining for BECN1 and GPX4; the endogenous peroxidase was inactivated by 3% H2O2 for 30 min. Next, the liver slides were immersed into preheated antigen retrieval solution (0.01 M, pH 6.0, citrate buffer) for 30 min. After blocking with 5% BSA, the liver slides were probed with primary antibody against BECN1 (Proteintech) and GPX4 (Abcam) overnight at 4 °C and incubated with biotin-conjugated anti-rabbit IgG and streptavidin-biotin. Finally, liver tissue sections were visualized with DAB Horseradish Peroxidase Color Development Kit (Boster, Wuhan, China) and counterstained with hematoxylin. Images were acquired using a pathological section scanner (3DHISTECH, Hungary).

The tissue blocks sampled from 116 cases of CRC were fixed with 4% formaldehyde, conventionally dehydrated before embedding in paraffin and sliced into 4-µm sections Immunohistochemical of CD163, CD4 and CXCR5 were performed using the EnVision two-step method on the Dako Omnis automated immunohistochemical stainer. All target antigens were prepared with high-pressure sodium citrate buffer at pH 6.0. PBS was used in place of the primary antibody for a blank control, and already-known positive cases were used as positive controls. The specific steps were carried out according to the kit manufacturers instructions. Rabbit anti-CD4 antibody, mouse anti-CD163 antibody and mouse anti-CXCR5 antibody were provided by Abcam. Immunohistochemical sections were scanned by the pathological section scanner (3DHISTECH). Five 400-fold visual fields were randomly selected, and positive cells were counted with Image pro plus 6.0 software to measure the average value.