Horac080pg01

Tissue microarray of HNSCC specimens (HOraC080PG01) was obtained from Shanghai Outdo Biotech Company (Shanghai, China) and used to validate the expression of CALR in HNSCC. Immunohistochemistry was then performed using the staining cycles as follows. In brief, formalin-fixed, paraffin-embedded HNSCC tissue sections were deparaffinized and then underwent microwave treatment in citrate for antigen retrieval. Then, they were blocked and incubated overnight with rabbit anti-Calreticulin antibody (1:500, ab92516, Abcam). Goat anti-rabbit IgG (Alexa Fluor 488, ab150077, Abcam) was used as the secondary antibody at 1/1000 dilution. DAPI was used as nuclear counterstain.

At the Hospital of Stomatology, Sun Yat-Sen University, we collected 91 oral squamous cell carcinoma (OSCC) tissue samples (including 17 highly differentiated samples, 41 moderately differentiated samples and 33 poorly differentiated samples) and 10 adjacent non-tumor tissue (ANCT) samples between September 2016 and January 2020. Another 9 normal oral tissues were selected from oral cancer tissue chip (HOraC080PG01, Shanghai Outdo Biotech Co, Shanghai, China). The cancer patients were selected based on their clinical and pathological information. Patients who had undergone preoperative radiotherapy or chemotherapy were excluded. We took two identical tissue samples from each patient. One was stored at − 80 ℃ for RNA extraction, and the other was fixed in formalin and embedded in paraffin for immunohistochemical analysis. All patients signed informed consent forms, and the study was approved and supervised by the Institutional Research Ethics Committee of the Hospital of Stomatology, Sun Yat-Sen University.