Aqueous mounting solution

Expression of CD4+ lymphocytes was detected by immunohistochemical analysis using the anti-CD4+ antibody. The skin tissues were rehydrated. After a microwave treatment, the sections were treated with 3% hydrogen peroxide in PBS for 15 min to inhibit endogenous peroxidase activity of blood cells. The skin sections were blocked with 5% bovine serum albumin (BSA) in PBS for 1 h, at room temperature. Skin sections were incubated with mouse monoclonal CD4+ antibody overnight at 4 °C and subsequently incubated with secondary biotinylated anti-rabbit IgG for 1 h at room temperature. Sections were treated with avidin-biotin HRP complex (Vectastain ABC kit, Vector Labs, Burlingame, CA, USA) for 30 min at 4 °C and finally stained with diaminobenzidine tetrachloride (DAB) as a substrate. The slides were mounted with an aqueous mounting solution (DAKO, Glostrup, Denmark) and cover-slipped. All the sections were analyzed using an Olympus microscope and images were captured using a digital video camera.

Differentiated ES cells were fixed with phosphate-buffered saline 4% paraformaldehyde (PBS-PFA) (Santa Cruz), treated for 1 h with PBS 0.1% Triton X100 (Merck Life Science) containing 5% donkey serum (Dako North America Inc) and 5% BSA (Merck Life Science) and incubated with primary antibodies for 3 h at 37 °C in a moist chamber. The following primary antibodies were used: anti-βIII-tubulin 1:1000 diluted in PBS (Merck Life Science) and anti-VE-cadherin 1: 100 diluted in PBS (R&D Systems). After rinsing three times with PBS, cells were incubated with 555 AlexaFluor donkey anti-goat and 488 AlexaFluor donkey anti-mouse (Invitrogen, Thermofisher Scientific) at 5 µg/ml for 1 h. After rinsing three times with PBS, nuclei were stained with DAPI and slides were sealed with aqueous mounting solution (Dako North America Inc). Images were captured by using a Leica TCS SPE confocal laser-scanning microscope, analyzed with Leica Confocal Software (LCS; Leica Microsystems). To quantify VE-cadherin⁺ endothelial cells, sequential images, covering the entire surface of a chamber slide well (0.7 cm²), were acquired with a Leica AF6000LX workstation. The area occupied by endothelial cells was measured with ImageJ software by using the Angiogenesis Analyzer for ImageJ [25 ].

CD4+ lymphocytes were detected by immunohistochemical analysis using anti-CD4+ antibodies (Santa cruz biotechnology, Dallas, Texas, USA). After deparaffinization, the slides were rehydrated and antigen retrieval done by microwave treatment, they were treated with 3% hydrogen peroxide in PBS for 15 min to inhibit the endogenous peroxidase activity of blood cells. Following the hydrogen peroxide treatment, the sections were incubated with 5% bovine serum albumin (BSA) in PBS as a blocking reagent for 1 h at room temperature. The sections were then incubated with mouse monoclonal CD4+ antibodies (1:100 dilution) overnight at 4 °C. After washing with PBS, subsequently incubated with secondary biotinylated anti-rabbit IgG for 1 h at room temperature. The sections were treated with an avidin-biotin HRP complex (Vectastain ABC kit, Vector Labs, CA, USA) for 30 min at 4 °C and stained with diaminobenzidine (DAB) tetrachloride as a substrate. The slides were mounted in an aqueous mounting solution (DAKO, Glostrup, Denmark) and cover-slipped. All of the sections were analyzed using an Olympus microscope, and images were captured using a digital video camera.