Rat anti cd31 antibody

At postoperative day 28, the gastrocnemius was harvested, fixed in methanol, paraffin-embedded, and cross-sectioned (6μm) for histological evaluation. Sections were blocked with 10% donkey serum. Primary antibodies were diluted in PBS containing BSA. Sections were stained for CD31 using a rat anti-CD31 antibody (BD) and smooth-muscle α-actin (αSMA) using a rabbit anti-αSMA antibody (Sigma). For immunofluorescent detection, AlexaFluor-conjugated secondary antibodies were used (Invitrogen). Nuclei were stained with DAPI (Research Organics). Using fluorescent microscopy (Zeiss), CD31⁺ capillary forms and CD31⁺/αSMA⁺ arterioles were quantified in 3 distinct high-power fields (20x) from 3 independent sections in each animal.

Endothelial cell (EC) proliferation in mice lungs was determined by staining BrdU labeled lung ECs as previously described (Liu et al., 2019 (link)). Briefly, BrdU (i.p., 150 mg/kg, BW; Sigma B5002) was injected in mice 14 h before sacrifice. Mouse lung cryosections (5 m thick) were stained with mouse anti-BrdU antibody (1:25, BD Biosciences), rat Anti-CD31 antibody (1:50, BD Biosciences) and DAPI. EC number was analyzed by using ImageJ software (NIH). The number of ECs with positive staining of BrdU was normalized to the total number of ECs. In vitro EC proliferation was also assayed in cultured ECs using the proliferation kit obtained from Abcam Inc (ab126556, Cambridge, United Kingdom).

Mice were anesthetized with Avertin and were perfused with cold PBS and subsequently with 4% PFA in PBS solution. The brains were removed, post-fixed with 4% PFA for 24 h and transferred to 30% sucrose solution. Frozen brains were cut with a thickness of 30 μm in a cryostat. Slices were preserved in 30%PEG 30% sucrose PBS. The brain slices were washed three times with Tris-buffered saline (TBS) and were then blocked with TBS-blocking solution (1 % bovine serum albumin, 0.2 % skim milk, and 0.3 % Triton X-100 in TBS) for 1 h and incubated with the following primary antibodies in TBS-blocking solution overnight on a shaker at 4 °C. Rabbit anti-S1P2R antibody (1:100, Proteintech Group Inc., Chicago, IL), rat anti-CD31 antibody (1:100, BD Biosciences, Pharmingen, San Jose, CA) were used as primary antibodies. Sections were washed three times with Tris-buffered saline (TBS) and then they were incubated with donkey anti-rabbit IgG- Alexa-594, goat anti-rat IgG Alexa-488 (1:250, Life Technologies, Molecular Probes, Grand Island, NY). Brain slices were stained with 4′,6-diamidino-2-phenylindole (DAPI) for 7 minutes and were mounted onto slides. Samples were observed on an Zeiss LSM 510 Meta Confocal microscope (Carl Zeiss Inc.). Images were captured using Zen LE software (Carl Zeiss). The specificity of the S1PR2 antibody was confirmed by lack of signal in the S1pr2^−/− brain sections.