Anti p53 clone do7

We evaluated the expression of four MMR proteins and of p53 by IHC using an automated stainer (Ventana, Tucson, AZ, USA). Primary antibodies included anti-MLH1 (Clone G168-728), anti-MSH2 (Clone G219-1129), anti-MSH6 (Clone 44), and anti-PMS2 (Clone EPR3947), all purchased from Roche (Basel, Switzerland). These antibodies were selected based on a previous study that recommended their use in IHC as a sensitive method to detect tumors with MSI.24 (link) The anti-p53 (Clone DO-7) and anti-D2-40 antibodies (Item number M3619, Clone D2-40) were purchased from DAKO (Carpinteria, CA, USA). Omission of the primary antibody was used as negative controls. For MMR proteins, normal colonic mucosa and lymphocytes were used as positive internal controls. Complete loss of nuclear staining in tumor cells was considered loss of expression. The absence of expression of any of the four MMR proteins was defined as dMMR. Tumors with preserved expression of all MMR proteins were considered mismatch repair protein proficient (pMMR). Known p53-positive cases were used as positive external controls for p53 immunostaining. Cases with at least 10% of the tumor cells exhibiting nuclear p53 staining were considered positive.25 (link)

To identify whether p53 accumulation influenced response to AI treatment, we performed IHC assay. Anti-p53 (Clone DO-7, DAKO, Carpinteria, CA, USA) antibodies were used at 1:100 dilution with a 10-minute high-temperature antigen retrieval in citrate buffer (pH=6.0). Detection was by EnVision+ (DAKO) with diaminobenzidine chromogen as per routine protocol. Known positive and negative controls were used to ensure the quality control of staining.