Borg decloaker

Immunohistochemical analysis of the thymus, spleen, and lymph node was performed as described previously [11 (link)]. Tissues were fixed in neutral buffer containing 10% formalin, and slides were prepared for the analysis. The endogenous peroxidase activity of the samples was blocked with 3% hydrogen peroxidase, followed by pretreatment with Borg Decloaker (Biocare, Cat. no. BD1000 S-250) and blocked in Background Sniper solution (Biocare, Cat. no. BS966 H). After washing, the samples were incubated with primary antibodies (Supplementary Table 2). After incubation, the samples were washed, incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies, and then they were stained with hematoxylin and eosin (H&E) to provide the background.

Rostral inguinal mammary glands and shaven back skin were fixed in 4% paraformaldehyde and paraffin-embedded. Antigen retrieval was carried out on mounted sections using a Borg Decloaker (Biocare Medical) and the sections were subsequently stained as described before [42 (link)]. Controls were performed using the secondary antibody only. See Supplementary Table 1 for the primary antibodies.

Formalin-fixed paraffin-embedded LN tissues were cut into 5 μm sections, deparaffinized at 60°C, and serially washed in 100% xylene, 95% ethanol, 80% ethanol, and 70% ethanol before 100% diH₂O baths (Thermo Fisher Scientific). Antigen retrieval was performed for 15 minutes at 110°C in a Borg decloaker (Biocare Medical). Slides were then submerged in 1× PBS for cooling, followed by permeabilization/blocking for 1 hour in PBS (Thermo Fisher Scientific)/bovine albumin (Millipore Sigma)/Triton-X (Thermo Fisher Scientific). Slides were then stained overnight (4°C) with titrated concentrations of primary antibodies. Slides were washed (3 times, 15 minutes each in 1× PBS) and then stained with titrated concentrations of secondary antibodies for 2 hours at room temperature, followed by 3 more washes. Slides were then blocked with mouse and or goat serum for 1 hour, followed by the addition of titrated concentrations of fluorochrome-conjugated antibodies for 1 hour. Slides were then washed 3 times and stained with nuclear stain (JoPro/Thermo Fisher Scientific) for 15 minutes. Coverslips were then mounted using Fluoromont G (Thermo Fisher Scientific). Tissue sections were stained using JoPro, CD3, CD4, PD-1, CD20, Ki-67, and BCL6. Tfh cells were defined as CD3⁺CD4⁺PD1^hi.