HES1 (D6P2U) Rabbit mAb #11988

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Publication protocol

Tumours and control tissues such as tongue mucosa were carefully dissected from the mice and fixed in 10% buffered formalin overnight. The tissues were embedded in paraffin. The sections of tissues were deparaffinized, rehydrated and subjected to antigen retrieval by sodium citrate (pH 6.0) or ethylene diamine tetraacetic acid (EDTA) for 20 min. Endogenous peroxidase activity was blocked with 3% H2O2 followed by serum block. The sections were incubated with primary antibodies overnight. On the second day, the sections were reacted with appropriated secondary antibodies. The staining was performed by ABC kits (Vector). The following antibodies were used in this study: Notch1, HES1, PD1, TIM3 (Cell Signaling Technology), LAG3 (Abcam), CTLA4 (Santa Cruz). All immunohistochemically stained slides were scanned using an Aperio ScanScope CS scanner (Vista, CA). Percent positivity (total number of positive pixels/total number of pixel) of whole section were quantified using Aperio ScanScope quantification software (Version 9.1). Area of interest was selected either in the epithelial or the stromal area for quantification. The histoscore of calculated value as a percentage of different percent positive cells were performed using the formula (3+) × 3 + (2+) × 2 + (1+) × 1 as previously described.24 The threshold for scanning of different positive cells was set according to the standard controls slices provided by Aperio.

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