Vector immunodetection kit

A human OSCC tissue microarray, containing 50 OSCC and 10 normal oral mucosal tissues, was bought from Alenabio Co., Ltd. (China). Immunohistochemistry (IHC) was performed using the Vector immunodetection kit (Vector Laboratories, USA). Sections were incubated with monoclonal mouse anti-SRSF3 (clone 7B4, invitrogen) overnight at 4 °C. SRSF3 specific nuclear expression in cancer or normal mucosal epithelia was scored into 4 levels (0 = less than 10% positive cells, 1 = 10%–33% positive cells, 2 = 34%–66% positive cells, 3 = more than 66% positive cells). Two independent observers evaluated IHC results. Disagreements were resolved by re-evaluation and discussion.

Mice were anesthetized with isoflurane inhalation and perfused with heparin/PBS to remove blood. Brain samples were harvested and frozen in dry ice, then sectioned into 20 µm sections. The location of AVM lesions was first identified through staining 1 of every 10 sections with lectin (1∶200; Vector Laboratories). Sections containing dysplastic vessels were then co-stained with primary antibodies against CD31 (1∶50; Abcam) and CD68 (1∶50; AbD Serotec). Each protein was subsequently visualized using fluorescently labeled secondary antibodies: Alexa Fluor 488 goat anti-rabbit IgG (1∶500; Invitrogen) for CD31, and Alexa Fluor 594 goat anti-rat IgG (1∶500; Invitrogen) for CD68. ENG expression was identified by an antibody against ENG/CD105 (1∶50; BD Pharmingen) and visualized by a biotin-conjugated secondary antibody (anti-rat IgG, 1∶500; Vector Laboratories), using the standard ABC method (Vector immunodetection kit; Vector Laboratories). Prussian blue staining was performed according to the protocol provided by the company (Iron Stain Kit; Sigma-Aldrich).