Simple stain max po m

The sample tissues were fixed in 10% formalin and were embedded in paraffin (FFPE). Four micrometer thick sections were prepared and were subjected to standard hematoxylin and eosin (H&E) staining or immunohistochemistry. After deparaffinization and washing in PBS, endogenous peroxidase activity was inactivated by treatment with 0.3% H₂O₂ for 30 min. Primary anti-HLA class I-A, B, and C antibodies (Hokudo, Sapporo, Japan) were applied at a 1:500 dilution and were incubated for 60 min. Simple Stain MAX PO (M) (Nichirei Biosciences) was used for secondary staining and incubated for 1 h. After washing, the cells were visualized with DAB and H₂O₂.

Paraffin-embedded mouse lung tissue was sectioned at a thickness of 3 µm. Deparaffinized/rehydrated sections were autoclaved at 120 °C for 10 min in citrate buffer (pH 6.0) for antigen retrieval. Sections were blocked for endogenous peroxidase activity using 3% hydrogen peroxide in methanol for 10 min and exposed to a blocking solution (#03649-64, Nacalai Tesque) for 10 min before incubation overnight in a humidified chamber at 4 °C with primary antibodies or isotype control. Immune complexes were detected by incubation with Simple Stain MAX-PO (M) (#424131; Nichirei, Tokyo, Japan) or Simple Stain MAX-PO (R) (#414341, Nichirei) at room temperature for 1 h before applying chromogen detection using diaminobenzidine (#425011, DAB substrate kit, Nichirei). Counterstaining was performed with Mayer’s hematoxylin (#30002; Muto pure chemicals, Tokyo, Japan) before dehydration. Sections were clarified with xylene and mounted using Marinol (#4197193, Muto pure chemicals). Images were acquired with a BZ-X810 microscope (Keyence) and analyzed with Image J software.