Rat anti mouse cd31 antibody clone mec13

Mammary tumor tissues were harvested, snap-freezed in Tissue-Tek O.C.T. compound (Sakura), 5 μm cryostat sections prepared, fixed in methanol for 10 min at −20°C, sections dried at −20°C for 30 min followed by drying by hair dryer for 5 min at room temperature, and rehydrated in DPBS for 5 min. Rehydrated sections were blocked by incubation with 1% bovine serum albumin (BSA) diluted in DPBS for 30 min at room temperature. Sections were then incubated overnight with a purified rat anti-mouse CD31 antibody (clone MEC 13.3, BD Pharmingen; 1:50 dilution in 0.1% BSA/DPBS) at 4°C in a humidified chamber. Following three washes in DPBS+0.1% Tween 20 (5 min each), sections were incubated with goat anti-Rat IgG (H+L) cross-adsorbed secondary antibody conjugated with alexa fluor 488 (Invitrogen; 1:200 dilution in 0.1% BSA/DPBS) for 45 min at room temperature protected from light, followed by 3 times wash with DPBS+0.1% Tween 20, and nuclear counterstained with Fluoro-Gel II with DAPI (Electron Microscopy Sciences). Sections (3–5 random fields/tumor section, n = 4 or 5 mice/group) were imaged at 20x magnification with a Nikon fluorescent microscope. ImageJ software (NIH) was used to quantify the CD31⁺ areas. Scientific personnel involved in acquiring fluorescent images and ImageJ analysis was blinded to the treatments.

The corneal cauterization assay was performed, as previously described [28 (link)]. Eight-week-old female C57BL/6 mice were anesthetized with a mixture of xylazine (10 mg/kg) and ketamine (100 mg/kg). After application of a local anesthetic (Unicaïne, 0.4%) to the eye, the cornea was thermally cauterized using an ophthalmic cautery. One day after cauterization, drops of 10 µL of CXCL9(74-103) (100 µg/mL) or PBS were applied daily for 4 days. On day 5, the mice were sacrificed, and the corneas were removed. Fixation and blocking of whole-mounted corneas were performed in 70% (v/v) ethanol for 1 h and 3% (w/v) BSA in PBS for 1 h, respectively. To stain for blood vessels, the corneas were incubated with rat anti-mouse CD31 antibody (clone MEC13.3, BD Biosciences) overnight and AlexaFluor568 goat anti-rat secondary antibody (Cat No A-11077, Invitrogen) for 2 h. Corneas were flat mounted on microscope glasses overlaid with Prolong Gold antifade mounting medium and imaged using a Leica DMI6000 microscope (Leica Microsystems, Wetzlar, Germany). The cornea blood vessel area was quantified using Leica MM AF morphometric analysis software (Leica Microsystems) and expressed as the percentage of the total corneal area. Ethical approval for animal experiments was obtained from the Ethical Committee of KU Leuven (number LA1210604).