Texas red and fitc conjugated secondary antibodies

Immunofluorescence staining was performed on cell monolayer, 2 hours after the scratch/wounds was performed. Cells were fixed in 4% paraformaldehyde (Electron Microscopy Sciences) at room temperature for 15 minutes and permeabilized in 0.1% Triton X-100 (Sigma) for 5 minutes. After blocking with 5% horse serum (Vector Laboratories) for 1 hour, cells were incubated with Alexa Fluor® 488 mouse anti-GM130 antibody (12.5 μg/ml; BD Biosciences) or ICAM2 (0.56 μg/ml; Cell Signaling) for 1 hour. Filamentous actin was stained using Phalloidin conjugated to the fluorescent dye tetramethylrhodamine (TRITC) (1:40; Life Technologies). Samples were mounted using Prolong Gold with 4′,6-diamidino-2-phenylindole (DAPI) (Life Technologies) and imaged with a C2 confocal microscope (Nikon).
Whole mount staining was performed on fibrin gels upon overnight fixation in 4% paraformaldehyde at 4 °C. Immunofluorescence was performed using primary antibodies against Podocalyxin (10 μg/ml; R&D Systems), Collagen IV (5 μg/ml; eBioscience) followed by Texas Red and FITC -conjugated secondary antibodies (1:200, Vector Laboratories). Nuclei were stained with DAPI (4′,6-diamidino-2-phenylindole) (1:1000, Life Technologies) and imaged Nikon A1R LUN-V Inverted Microscope.

The tumors were collected, frozen in liquid nitrogen, and sectioned into 5 μm slices. Macrophages in the tumor sections were stained with anti-F4/80 and anti-CD206 antibodies (Abcam, Cambridge, UK), followed by appropriate fluorochrome-conjugated (Alexa Fluor 594 (Abcam) and FITC (Vector Laboratories, Burlingame, CA, USA) secondary antibodies. The cytotoxic T lymphocytes were stained with anti-CD8α (Abcam) and subsequently with Alexa Fluor 594-conjugated secondary antibodies (Abcam). For the immunohistochemical analyses of pericytes coverage, tumor blood vessels sections were incubated with anti-α-Smooth Muscle Actin and anti-CD31 antibodies (Abcam) and subsequently with Texas Red and FITC-conjugated secondary antibodies (Vector Laboratories) [13 (link)]. All the sections were mounted in VECTASHIELD Mounting Medium with DAPI (Vector Laboratories). Microscopic observations were performed using a LSM710 confocal microscope (Carl Zeiss Microscopy GmbH). The obtained confocal images were analyzed with ImageJ 1.48v (National Institutes of Health and the Laboratory for Optical and Computational Instrumentation, LOCI, University of Wisconsin, USA) and the results were expressed as the percentage of area [%].