Meca 79

Tissue microarrays (TMAs) were constructed for immunohistochemistry. The tissue cores (2 mm) from two representative areas were punched and placed into the recipient blocks using a manual TMA device (SuperBioChips Laboratories, Seoul, Korea). Immunohistochemistry was performed using a Ventana Benchmark Autostainer (Ventana Medical System, Tucson, AZ, USA). Briefly, the slides were de-paraffinized, and antigen retrieval was conducted using heat-induced (92 °C for 30 min) epitope retrieval with an MC1 solution (Ventana Medical System, Tucson, AZ, USA). Sections were incubated with primary antibodies: CD8 (1:100, C8/144B, Dako, Cambridge, UK), Foxp3 (1:100, 236A/E7, Abcam, Cambridge, UK), CD20 (1:600, L26, Dako, Cambridge, UK), and MECA-79 (1:200, Santa Cruz, Tucson, AZ, USA). The Ultraview Polymer Detection Kit (Ventana Medical System, Tucson, AZ, USA) was used for visualization, and the stained sections were counterstained with hematoxylin. Human tonsil tissue was used as the positive control tissue. A negative control was performed by replacing the primary antibody with normal serum.

Formalin-fixed paraffin-embedded tissues were sectioned (4 μm) and stained with hematoxylin and eosin (H&E) for histological analysis or used for immunohistochemistry (IHC). For IHC, endogenous peroxidases were inactivated by 3% hydrogen peroxide and nonspecific signals were blocked by 1% BSA. Sections were incubated with the primary antibody: CD20 (1:200, #MA5-13141, Invitrogen), CD3 (1:200, #ab16669, Abcam), MECA-79 (1:100, #sc-19602, Santa Cruz), CD21 (1:100, #sc-13135, Santa Cruz), CD23 (1:100, #MA5-14572, Invitrogen), TCL1A (1:500, #ab108978, Abcam), PAX5 (1:1000, #ab109443, Abcam), TNFRSF13C (1:300, #ab168389, Abcam), CD79A (1:200, #ab79414, Abcam) at 4°C overnight, HRP-conjugated secondary antibody at 37°C for 1 h, and subsequently stained with DAB substrate. Counterstaining was performed with hematoxylin and mounted with a mounting medium.

Uteri were fixed overnight in 4% paraformaldehyde (PFD) before being processed and dehydrated through increasing concentrations of ethanol, cleared in chloroform and blocked in a paraffin wax. Tissues were cut into 5 µm sections, deparaffinized in xylene, rehydrated in reducing concentrations of ethanol. Tri-sodium citrate (pH 6.0) was used for antigen retrieval, while 1% H₂O₂ in PBS was used to neutralize the endogenous peroxidase. Sections were blocked in 5% BSA for non-specific binding, prior to incubation with rat monoclonal primary antibody: MECA-79 (200 μg/0.1ml, 1:500) (Santa Cruz, SC 19602) in 5% BSA at room temperature for 1 h. After rinsing with PBS, sections were incubated with biotinylated secondary antibody for 30 min at room temperature and were then exposed to avidin and biotinylated HRP complex (Santa Cruz, California, USA) in PBS for another 30 min. The site of antibody binding was visualized by DAB (Diaminobenzidine HCl) (Santa Cruz, California, USA) which gave dark brown stains. Sections were counterstained with hematoxylin for nuclear staining. In this experiment, non-immune normal donkey serum was used as a negative control where no staining was observed.