Intact GBM-bearing brains were sliced coronally (1 mm slice thickness) and embedded in paraffin. 5 μm-thick continuous sections were cut for hematoxylin and eosin (H&E) staining and immunohistochemistry (IHC) staining, followed by high-resolution Raman imaging on paraffin blocks. IHC staining was performed on the Discovery XT biomarker platform (Ventana, Tucson, AZ) as previously described 30 . Antibodies for OLIG2 (1:300, AB9610, Millipore, Temecula, CA), polyethylene glycol (1:100, ab51257, Abcam, Cambridge, MA), ITGB3 (1:100, 13166, Cell Signaling, Danvers, MA), ITGAV (1:2000, ab76609, Abcam), HA-tag (1:200, 11867423001, Roche, San Francisco, CA), IBA1 (1:600, 019-19741, Wako, Richmond, VA), NOS3 (1:200, 610296, BD Biosciences, Franklin Lakes, NJ) and ACTA2 (1:350, M0851, DAKO, Carpinteria, CA) were used as the primary antibodies. The slides were digitally scanned with Pannoramic Flash (3DHistech, Hungary) and relevant tissue areas were exported into tiff format. Quantification of Olig2 was performed using ImageJ/FIJI (NIH). Color deconvolution algorithm was used to determine the area of positive signal, which was normalized to tissue area.
Ab76609
Manufactured by Abcam
Ab76609 is a high-quality anti-GFP antibody produced by Abcam. It is designed for the detection of green fluorescent protein (GFP) in various applications. The antibody has been validated for use in western blotting, immunohistochemistry, and immunocytochemistry.
Automatically generated - may contain errors
2 protocols using ab76609
1
Multimodal Imaging of Glioblastoma
2
Quantification of Tumor Angiogenesis Markers
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Three VX2 tumor models in the TG and NTG were sacrificed on day 1 after the imaging, and the tumors were resected for H&E and immunohistochemical staining. The tumor tissues were fixed in 10% formalin solution and then embedded in paraffin to prepare serial sections. Immunohistochemical staining was performed by the streptavidin-biotin complex (SABC) method following deparaffinization and rehydration and treated with antigen retrieval. The primary antibodies used were a mouse monoclonal CD31 antibody (ab9498, Abcam Inc., Cambridge, MA) at a 1:50 dilution and a rabbit polyclonal anti-integrin α v antibody (ab76609, Abcam Inc.) at a 1:200 dilution and were reacted overnight at 4 C. Subsequently, the biotinylated secondary antibody and SABC complex were applied and diaminobenzidine (DAB) substrate was used to stain the sections. The sections were then treated with hematoxylin-eosin (H&E) and mounted.
The images in 2,088 × 1,550 pixels were acquired with an Olympus BX51 microscope and processed by Leica QWin version 3.4.0 software. The slides were initially screened at low power to identify the areas with the largest numbers of stained hot spots. The mean values of immunostained areas in pixels and the percentage of immunostained area per microscopic field were calculated in 200× magnification in the four most immunostained areas.
The images in 2,088 × 1,550 pixels were acquired with an Olympus BX51 microscope and processed by Leica QWin version 3.4.0 software. The slides were initially screened at low power to identify the areas with the largest numbers of stained hot spots. The mean values of immunostained areas in pixels and the percentage of immunostained area per microscopic field were calculated in 200× magnification in the four most immunostained areas.
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