Sniper universal blocking sera

Tumor from all groups of mice were sectioned for histological studies. The tissue samples were fixed in 10% formalin and embedded in paraffin. The sections (5 μm) were cut using microtome, stained with hematoxylin and eosin, and slides were assessed using microscope (Leica microsystems, Germany) using at original magnification 10× and processed in Adobe Photoshop. For the immunofluorescence study, paraffin embedded sections were deparaffinized with xylene followed by rehydration with descending alcohol series. Slides were steamed with Reveal Decloaker (Biocare Medical), to minimize background staining, Sniper Universal Blocking Sera (Biocare Medical) was used throughout the protocol. Primary antibodies for Ki67 (Invitrogen), cd133 (Invitrogen) were diluted according to the manufacturer’s instruction and incubated overnight at 4 °C. Subsequently, Alexa Fluor-conjugated secondary antibody (Invitrogen) were used for visualizing. Slides were counterstained with DAPI and observed in a Leica fluorescent microscope. Negative control slides were used to discriminate nonspecific staining.

For immunohistochemistry, paraffin tissue sections were deparaffinized in xylenes and hydrated through graded ethanol. Hematoxylin and Eosin (H&E) staining were used for evaluation of histological features. Slides were steamed with Reveal Decloaker pH9.0 (Biocare Medical) for antigen retrieval of GLUT1 antibody (Sigma) and CD133 antibody. Sniper Universal Blocking Sera (Biocare Medical) were used throughout the protocol. Primary antibodies were diluted according to vendor's instruction and incubated overnight at 4°C. The primary antibody was omitted for the negative controls. For immunofluorescence, fluorescent antibody conjugates were used after primary antibody staining. Slides were counterstained with DAPI and visualized in a Nikon fluorescent microscope. Tissue samples were incubated with mouse IgG1 isotype controls (BD Biosciences) and did not demonstrate any specific staining.