Benchmark ultra autostainer

Samples from 70 patients were stained via the EnVision two-step immunohistochemical technique, using a Leica Benchmark-ULTRA Autostainer, for four antigens, namely, Notch3, Jagged1, Jagged2, and DLL4. Notch3 was positive in the cytoplasm and cell membrane; DLL4 was positive in the cell membrane; and Jagged1–2 were positive in the cytoplasm in a spotty manner. Positive standard: (1) strong staining; (2) percentage of positive tumor cells >75%. Sections of human breast cancer submitted to the same immunohistochemical protocol were used as a positive control.

Samples from 20 patients were stained via the EnVision two-step immunohistochemical technique, using a Leica Benchmark-ULTRA Autostainer for MAFB antigens. The subcellular location of MAFB is mainly in the nucleus, but a small part is still located in the plasma membrane, Golgi apparatus, lysosome, endoplasmic reticulum, and mitochondrion. IHC analysis was performed to detect the protein expression level with the MAFB antibody (Cell Signaling Technology, #30919 S, 1:200). Two independent pathologists, who were unknown of the patient clinical information and histopathological features of the samples, were responsible for reviewing and scoring the degree of immunostaining separately. Human normal muscle tissue was selected to perform the same immunohistochemical protocol and was used as a negative control. Similarly, osteosarcoma tissue was acquired from the nude mice model to perform the same immunohistochemical protocol for staining Ki-67. The patients' details of immunohistochemistry specimens used in this study are shown in Table S1.