Rat anti mouse cd31

For the ex vivo detection of vascular permeability, WEHI-164 or CT-26 bearing BALB/c mice were injected i.v. with PBS, L19-mTNF (250 μg/Kg) or with GSK’963 (2 mg/Kg) followed by L19-mTNF (250 μg/Kg; 30 min later). After 24 hours, 10 mg/Kg of Hoechst 33342 (Thermo Scientific) was injected into the tail vein, and animals were euthanized after 1 min. Tumor, kidney and liver were collected, embedded in cryoembedding medium (Thermo Scientific) and snap frozen in liquid nitrogen. Cryo-sections of 10 μm were fixed in ice-cold acetone for 10 min and blocked for 1 hour in FBS (20% solution in PBS). The vessels were stained with a rat anti-mouse CD31 (BD Pharmigen; 1:100) and with a donkey anti-rat Alexa Fluor 594 as a secondary antibody (Invitrogen; 1:500). Slides were mounted with fluorescent mounting medium (Dako, Glostrup, Denmark) and analyzed with Leica TIRF epi-fluorescence microscope (Scientific Center for Optical and Electron Microscopy ScopeM, ETH, Switzerland). Pictures representing a sample overview were obtained by stitching electronically adjacent regions.

The skin tissues were collected and fixed overnight with 4% paraformaldehyde following by washing with PBS and soaking in 10% sucrose for 2 h and then 20% sucrose overnight. The wound tissues were embedded in O.C.T compound (Sakura Finetek, Tokyo, Japan) and frozen in liquid nitrogen to make frozen blocks of samples. The frozen wound tissue sections were mounted, stained with hematoxylin and eosin (Wako), and observed under a microscope (Olympus, Kyoto, Japan) to assess the tissue structure.
Immunohistochemical staining was used to analyze the role of AT-MSCs in recruiting inflammatory cells and forming vessels at wound sites. The inflammatory cells recruited to the ischemic area were examined by immunohistochemical staining of the wound tissues with rat anti-mouse CD45 (553078; BD Pharmigen Inc., San Diego, CA, USA), and rat anti-mouse Mac1 (rat anti-mouse CD11b 550282; BD Pharmigen). The neovascularization was analyzed by immunohistochemical staining of wound tissues with rat anti-mouse CD31 (553370; BD Pharmigen), according to the manufacturer’s instructions. The numbers of positive cells were counted in 10 randomly selected fields, and the average was determined.

Mouse brains (n = four for control wild type, R26^{GT-Map2k1-GFP/+} or Tg-Cdh5CreER^+/− and n = 4 for mutant R26^{GT-Map2k1-GFP/+}; Tg-Cdh5CreER^+/−) were fixed in formalin, embedded in paraffin, sectioned (seven µm), and hematoxylin/eosin stained. Tissue undergoing immunostaining were flash frozen in OCT and stored at − 80 °C. Seven µm cryo-sections were cut. Primary antibodies used: rabbit anti-mouse COL15A1 (Gift from Dr. Karppinen, University of Oulu, Finland) (1/500) and rat anti-mouse CD31 (1/500) (BD Pharmigen: 553370). Secondary antibodies used: donkey anti-mouse IgG (H + L) Alexa Fluor 488 (Abcam 150105, 1/200); donkey anti-rabbit IgG (H + L) rhodamine red-X (1/500) (Jackson ImmunoResearch 712-295-150). Sections were mounted with DAPI Fluoromount-G (SouthernBiotech). Images of sections were obtained using an Eclipse 80i microscope (Nikon) with a Spot model 25.4 camera and Spot 5.2 software (Diagnostic Instruments). Confocal images were captured using a Zeiss LSM 800 confocal microscope and Zen Blue software version 2.5.