Ab150440

The FFPE tissue sections were deparaffinized to water using graded ethanol-dimethylbenzene, employed in ethylenediaminetetraacetic acid (EDTA) buffer (pH 8.0, ZHHC, PI001) for 15 min to recover the antigen, incubated in Triton X-100 (ZHHC, PI024) for 5 min to implement a permeable membrane, and sealed with bovine serum albumin (BSA). To label the T_H1, T_H2, and T_reg subsets of the TIME in TNBC, sections were incubated overnight at 4 °C with primary antibodies against T-bet (Abcam, ab150440, 1:30), GATA-3 (Abcam, ab199428, 1:30), and CD25 (Abcam, ab231441, 1:50) and CD4 (Proteintech, 67786-1-Ig, 1:200). The sections were washed with phosphate-buffered saline (PBS) and incubated with the secondary antibody (Proteintech, SA00003-1 and SA00009-2, 1:50) at room temperature for 60 min. The nuclei were stained with 4,6-diamidino-2-phenylindole (DAPI) (ZHHC, CD110) for 3 min. The stained cells were observed, and images were acquired by a confocal laser scanning microscope (Leica Company) in at least 10 fields per section. For Masson’s trichrome staining, the deparaffinized sections were analyzed with a Masson’s trichrome stain kit (Solarbio, G1340) and were observed, and images were acquired by a Leica scanning optical microscope after dehydration, clearing, and mounting.

FFPE blocks of kidney allograft biopsies from transplant patients were cut (4 μm), then underwent sequential rehydration and antigen retrieval in citrate pH 6 solution buffer. The sections were incubated overnight with anti-CD20 (mouse, M0755, Dako), anti-CD27 (armenian hamster, ab219779, Abcam), and anti–T-bet (rabbit, ab150440, Abcam) antibodies. After washing, the sections were incubated 30 minutes with secondary anti-mouse (AF488, Dako), anti–armenian hamster (AF568, Abcam), and anti-rabbit (Cy5, Abcam) antibodies. Nuclei were counterstained with DAPI, and slides were mounted using ProLong medium (Thermo Fisher Scientific). Sections were also stained with hematoxylin and eosin for histological evaluation. Each experiment was performed concomitantly with a positive control (section from kidney allograft removed due to incurable ABMR or human spleen) and a negative control (section from kidney allograft incubated with secondary antibody without primary antibody). Images were acquired on a Zeiss Axio Scan instrument. We used the number of CD20⁺CD27⁺T-bet⁺ (triple-positive) cells per 10 consecutive high-power fields for cell quantification.