Ab64088

For histological analysis, inguinal lymph nodes were collected at 7 weeks after the first vaccination and fixed in 4% paraformaldehyde. Fixed tissues were embedded in paraffin, cut and stained with hematoxylin and eosin (HE).
For immunohistochemical analysis of CD3 (T cells), CD20 (B cells), and Bcl6 (follicular B cells and T cells), the deparaffinized sections were heated for antigen retrieval. After blocking with Antibody Diluent with Background Reducing Components (Dako), the sections were incubated with primary antibodies (CD3: ab16669 abcam, CD20: ab64088 abcam, Bcl6: ab272859 abcam) for overnight at 4 °C. After washing, the sections were treated with HRP-labelled pokymer anti-rabbit (Dako Envision system, Dako) for 30 min at RT, and developed with the ImmPACT DAB system (Vector) for 5 min. The sections were counterstained with Mayer’s hematoxylin. Stained tissue sections were observed under a BZ-X810 microscope (Keyence).

H&E staining and Immunohistochemistry were performed as previously described (15 (link), 16 (link)). Briefly, tumor specimens were first fixed with 10% formalin for 24 h and 70% ethanol for 12 h prior to embedding in paraffin and sectioning. Tissue sections were then deparaffinized in xylene and rehydrated in graded ethanol.
For H&E staining, after staining with hematoxylin for 5 minutes, sections were stained with eosin solution for 30 seconds. Next, sections were dehydrated and mounted using neutral gum.
For IHC, heat-induced antigen unmasking was performed in a 10mM citrate buffer and then blocked with 1% BSA for 1 h at room temperature. Sections were incubated with primary antibodies at 4°C overnight at an ideal dilution in a humidified chamber. On the next day, sections were incubated with secondary antibodies for 30 minutes at room temperature (Zsbio, pv8000), followed by detection using the DAB detection kit (OriGene Technologies, ZLI-9017). The following primary antibodies were used: anti-Ki67 (Abcam, ab15580; 1:100), anti-F4/80 (Cst,70076; 1:200), anti-CD20 (Abcam, ab64088; 1:100), anti-CD4(Abcam, ab183685; 1:100), anti-CD8 (Abcam, ab228965; 1:100), anti-Collagen type IV (Affinity, af0510; 1:100), anti-CK7 (PTG, 17514-1-Ap; 1:100), anti-CD34 (Abcam, ab81289; 1:100), and anti-Ly6G (Invitrogen, 2335909; 1:100).

Tumor sections with 4‐μm thickness were fixed by formalin and then embedded by paraffin, which were subsequently stained with CD3, CD20, and PD‐1. Immunohistochemistry (IHC) was performed as previously described.²² The sections were incubated with the detection antibody (CD3 [Abcam, ab16669, China], CD20 [Abcam, ab64088, China], and PD‐1 [Abcam, ab52587, China]) for 1 h at 37°C. Afterward, the sections were incubated with secondary antibody for 30 min and followed by DAB (DAKO Liquid DAB) staining for 5 min. Finally, hematoxylin counterstain was used to show the cellular nucleus. After the sections were sealed with neutral balsam, representative images were taken and analyzed.

The paraffin-embedded tissue slices (thickness 3–4 μm) were dewaxed, rehydrated by continuous immersion in ethanol and sealed with a peroxidase sealing solution for 5 min. The primary antibodies (anti-LC3-B, ab48394; anti-mTOR, ab134903; anti-CD3, ab135372; anti-CD14, ab221678; anti-CD20, ab64088; anti-CD68, ab125212; 1:250, Abcam) were added and incubated with the sections, and the sections were stained with biotin-linked secondary antibodies before being treated with streptavidin (K3461, DACCO Cytomation). Subsequently, the slides were treated with an AEC solution at room temperature for 10 min, and images were taken with a fluorescence microscope (Nikon, Tokyo, Japan).