Axiovert 200 base microscope

For immunofluorescent staining of CD31, CD31 rat anti-mouse mAb (Life Technologies, Inc.), and Alexa Fluor® 594 goat anti-rabbit IgG (H+L) antibody (Life Technologies, Inc.) were used as primary and secondary antibodies, respectively. Briefly, sections were blocked with 10% goat bovine serum albumin for 30 min, followed by overnight incubation at 4 °C with the primary antibody and 45 min at 4 °C with the secondary antibody. After each step slides were washed with PBS. Fluorescent images were taken with a Zeiss Axiovert 200 base microscope.

Tumor-bearing mice were i.v. injected with 10 μg (~ 72 nmol) Dex1-Cy5.5 (200 μL of 0.36 mM in saline solution). One hour later, mice were sacrificed, and the brains were harvested, frozen, and cryosectioned at a thickness of 5 μm. Tissue slides were counterstained with DAPI (Sigma). Images of the brain section were acquired using a Zeiss Axiovert 200 base microscope.

Tumors were excised at 30 minutes after a single injection of FITC-10KD dextran (375 mg/kg bw, n=1) or at 1 hour after a single injection FITC-10KD dextran (375 mg/kg bw, n=1) were fixed in PFA and processed for histology. For immunofluorescent staining of CD31 and CD31 rat anti-rabbit mAb (Life Technologies, Inc.) and Alexa Fluor® 594 goat anti-rabbit IgG (H+L chain) antibody (Life Technologies, Inc.) were used as primary and secondary antibodies, respectively. Sections were blocked with 10% goat bovine serum albumin for 30 min, followed by overnight incubation at 4 °C with the primary antibody and 45 min at 4 °C with the secondary antibody. After each step, slides were washed with PBS. Fluorescent images were taken with a Zeiss Axiovert 200 base microscope.