Axioplan 2 confocal microscope

Tumours (n=4) from each MCF-7 xenograft treatment group were sectioned (7 μm) from various positions across the tumours, deparaffinised in two changes of Histolene, followed by rehydration in decreasing concentrations of methanol (from 100% to 50%). Slides were then subjected to heat-induced epitope retrieval for 30 min in 10 mM citrate buffer pH 6 and cooled for 30 min. Following washing, slides were incubated 30 min in blocking solution (1% BSA, 10% horse serum in PBS). Next, the tumour sections were incubated overnight at 4 °C with anti-endomucin antibody (Santa Cruz Biotechnology Inc.), 1 : 500 dilution in blocking solution. Subsequently, the tissues were incubated in 1 : 800 anti-rabbit Cy5-labelled secondary antibodies (Jackson Immuno Research) at room temperature for 2 h. Tissues were then washed, mounted and stained with DAPI using ProLong Gold antifade reagent.
Representative images were captured on a Zeiss Axioplan 2 confocal microscope (North Ryde, NSW, Australia) at a magnification of × 200. To quantitate the images of five different sectors of each tumour, ImageJ (version 1.47c) was used to count the number of lumina and blood vessels for each experimental condition. The data were expressed as the average number of blood vessels and lumina per experimental condition.

Astrocytes primary culture was obtained from the cerebral cortex of neonatal (3–7 days old) albino rats of the Wistar strain (Rattus norvegicus) according to the previously established protocol (15). Rats were provided by the Universidad Autónoma Metropolitana-Iztapalapa animal facility. A total of 24 neonatal rats were used for the study and three animals were used for each culture. Cells were grown in MEM medium supplemented with 10% fetal bovine serum (BSA), .11% glutamine, .15% glucose, and .1% penicillin-streptomycin and were subsequently incubated at 37°C and 5% CO2. The medium was replaced every third day. Cells were divided when they reached confluency and reseeded continuously. All procedures with animals were strictly carried out in accordance with the guide for handling and care of laboratory animals of the National Institutes of Health, and the official Mexican standard for handling animals NOM: 062-ZOO-1999.
The glial fibrillary acidic protein (GFAP) detection by immunocytochemistry was performed to confirm culture purity as described before.^{15 (link)} Single plane images were obtained in a LSM-META-Zeiss Axioplan-2 confocal microscope at 40X magnification; the 405 nm Laser Diode was used for DAPI and the Ar/ML 458/488/514 nm for the ALEXA 594. Images were analyzed using the ZEN 2010 version 6.0 program from Carl Zeiss.