Trisedta antigen retrieval buffer

Sections were deparaffinized in successive incubations with xylene and decreasing concentrations (100, 95, 75, 50, 0%) of EtOH in ddH2O. Antigen retrieval utilized Abcam 100× TrisEDTA Antigen Retrieval Buffer (pH=9) heated under high pressure and washed in phosphate buffered saline (PBS)+0.1% Tween 20. Sections were blocked for 30 min with 10% goat serum in 1×PBS+0.1% Tween 20 before staining. Primary antibodies were used at a concentration of 1:100 (MHC-II) or 1:150 (CD8, TCF-1) and incubated for 1 hour at room temperature. Secondary antibodies were used at a concentration of 1:250 (A488, A568) or 1:500 (A647) and incubated for 30 min at room temperature. Details about antibodies used are listed in table 2. Sections were counterstained with 4’,6-diamidino-2-pehnylindole (DAPI) according to manufacturer instructions (Thermo Fisher). IF images were collected using a Zeiss Z.1 Slide Scanner equipped with a Colibri 7 Flexible Light Source, and Zeiss ZenBlue software was used for post-acquisition image processing. CellProfiler42 43 (link) and custom R and python scripts were used for image analysis, as previously described,44 (link) to determine the xy coordinates of cells within tissue slices, measure fluorescence intensity within each cell, calculate cellular density, and create spatial maps of features within the tissue.