Goat anti rat alexa 594

Immune-fluorescence staining for CD8⁺ T cells in the tumors of WT, BLT1^−/− and CXCR3^−/− mice was analyzed using Nikon-A1R Confocal microscope. Tumors were embedded in OCT medium and snap frozen in liquid nitrogen and later cut into 5µm sections using a cryostat. Sections were fixed using ice-cold acetone and then blocked using 1X PBS supplemented with 3% BSA and 5% goat serum for 1 hr at RT. To stain for CD8⁺ T cells, the sections were incubated with rat anti-mouse CD8a Ab (BD Pharmingen) in 1X PBS + 3% BSA for 1hr at RT. After 3 washes with PBS, the sections were then incubated with the secondary Ab goat anti-rat Alexa 594 (2mg/ml, Invitrogen). After washing with PBS, the sections were mounted with Vectashield mounting medium containing DAPI (Vector Labs) and analyzed at 200X magnification. A minimum of 4 fields for each tumor section was analyzed.

Tumors were harvested from mice and frozen in OCT freezing medium (Tissue-Tek), then sectioned for staining as previously described (26 (link)). Primary and secondary antibody was rat anti-mouse CD31 antibody (1:50, BD Biosciences) and Alexa 594 goat anti-rat (1:2000, Invitrogen), respectively. Five random 10X magnification pictures were taken of each slide and the area of CD31⁺ structures, visible lumens, total vessels, and vessels ≥100µm were counted. Images were taken with a 10× or 20× magnification objective lens and with a digital camera AxioCAM HRc (Zeiss, Thornwood, CT) mounted on Zeiss Imager M1 Axio using Zeiss AxioVision Acquisition software (version 4.5).

LuEC were plated in triplicate at 5 × 10³ cells per well in 0.1% gelatin coated 24-well tissue culture plates. Cells were counted by a coulter counter (Beckman). For 5-bromo-2-deoxyuridine (BrdU) labeling, 2.5 × 10³ LuEC were plated onto 0.1% gelatin-coated glass coverslips and then serum starved for 24 hours, then incubated with drug treatment and then pulsed with 10µM BrdU (BD Biosciences) for 1.5 hours. Cells were then fixed with 4% paraformaldehyde, permeabilized with 0.1% TBST, and denatured with 2N HCL. Endothelial cells were stained with anti-BrdU mouse antibody (1:50, Dako). Secondary antibody was Alexa 594 goat anti-rat (1:2000, Invitrogen) and nuclei were identified with Hoechst33342 (1:1000, Invitrogen). Seven random 10X magnification pictures were taken of each slide using 10× or 20× magnification objective lens with a digital camera AxioCAM HRc (Zeiss, Thornwood, CT), BrdU positive cells and total cell number were counted. In situ terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling (TUNEL) labeling was performed using the DeadEnd Fluorometric TUNEL System (Promega, Madison, WI) according to manufacturer's instructions. Flow cytometry of TUNEL stained cells was performed on a FACS Canto flow cytometer (BD Biosciences) and analyzed with FlowJo Software (TreeStar, Ashland, OR).