Anti vwf

Testis samples were immersed in lysed buffer and centrifuged at 13,000 rpm for 15 min at 4°C. Bicinchoninic Acid (BCA) Kit (Pierce, Rockford, IL, USA) was used to measure the protein concentration in the supernatants with steps according to the manufacturer's protocol. Total protein (50 μg) was added to each lane onto 10% SDS-polyacrylamide gels. After electrophoresis, we used Tris-buffered saline containing 0.1% Tween-20 to wash the polyvinylidene fluoride (PVDF) membranes (Millipore, Billerica, MA, USA) and then incubated them with primary antibody: anti-Bcl-2 (diluted 1 : 1000, Cell Signaling Technology, Beverly, MA, USA), anti-Bax (diluted 1 : 1000, Cell Signaling Technology, Beverly, MA, USA), anti-vWF (diluted 1 : 1000, Abcam, Britain), and anti-VEGF (diluted 1 : 1000, Abcam, Britain) at 4°C overnight. Secondary antibody (1 : 5000, Pierce, Rockford, IL, USA) was added to the membrane for 2-hour incubation. Protein was visualized with Immobilon Western Chemiluminescent HRP Substrate (Millipore, Millipore Corp., Bedford, USA). Band intensity was quantified by densitometry using the Image J software (National Institutes of Health, Bethesda, MD, USA).

For immunohistochemistry, the tissue was fixed in 4% paraformaldehyde overnight. Following deparaffinization and rehydration, sections (5 mm) were rinsed for 6 min using PBS. Endogenous peroxidase activity was quenched using 0.3% H₂O₂ for 10 min. After 6 min of washing with PBS, the tissue was blocked using 3% bovine serum albumin (BSA) for 30 min and then incubated with anti-nNOS (Santa Cruz Biotechnology Inc., USA; 1:100), anti-ɑ-smooth muscle antigen (Abcam Inc., Hong Kong, China; 1:400), or anti-vWF (Abcam Inc., Hong Kong, China, 1:800) at 4 °C overnight. Sections were then incubated with goat anti-rabbit secondary antibodies (Dako, Glostrup, Denmark; 1:100) for 2 h at room temperature and then counterstained with hematoxylin. Sections incubated without primary antibodies were used as negative controls. Images were captured using a Nikon microscope with a Spot RT color digital camera and digital histomorphometric analysis was performed using Image-Pro Plus 6.0 software.

We performed double immunostaining with anti-pERK1/2 and anti-von Willebrand factor (vWF; endothelial specific marker) antibodies to localize ERK1/2 activation in the lung endothelial cells. Fresh frozen lung tissues were incubated with 7.5% normal donkey serum for 1 h to block nonspecific protein binding, after which they were incubated overnight at 4 °C with the following primary antibodies: anti-phospho-ERK1/2 (Cell Signaling, Danvers, MA, USA; 4370, dilution 1:150) and anti-vWF (Abcam, Cambridge, MA, USA; ab11713, dilution 1:50). The primary anti-pERK1/2 and anti-vWF antibodies were detected by incubation with fluorescein-conjugated donkey anti-rabbit (Alexa Fluor 488, dilution 1:200) and donkey anti-sheep (Alexa Fluor 633, dilution 1:200) secondary antibodies, respectively. An indirect TUNEL assay was used to detect apoptosis using the ApopTag Fluorescein In Situ Apoptosis detection kit (MilliporeSigma, St. Louis, MO, USA; S7110), as per the manufacturer’s recommendations. The localization of the apoptotic process in the endothelial cells was determined using vWF antibodies, as described above. All the slides were counterstained with 4′,6-diamidino-2-phenylindole (DAPI) and analyzed by confocal microscopy. The observers analyzing these slides were masked to the experimental conditions.

Whole-cell lysates were resolved on a denaturing 10% SDS-PAGE gel and subsequently transferred to polyvinylidene fluoride membranes via semidry transfer. After blocking the membrane at room temperature with 5% skim milk for 1 h, the membrane was incubated overnight at 4°C with anti-CD31 (1:1000, abcam), anti-CD34 (1:1000, abcam), anti-VWF (1:1000, abcam) antibodies. After incubation with peroxidase-conjugated secondary antibodies at a dilution of 1:2000 for 1 h and washed three times with PBS, the signals were visualized using enhanced chemiluminescence.

Cells and tissue sections treated as described in 2.3., and finally incubated overnight at 4°C with the following primary antibodies: anti-Nestin (1:500, mouse IgG1; BD Biosciences, United States), anti-SOX2 (1:500, rabbit IgG; Abcam, United States), anti-MAP2 (1:500, rabbit IgG; Abcam, United States), anti-NeuN (1:800, mouse IgG; Abcam, United States), anti-GFAP (1:1,000, rabbit IgG; Cell Signal Technology, United States). Anti-BrdU (1:500, mouse IgG1; BD Biosciences, United States), anti-vWF (von Willebrand factor, 1:200, rabbit IgG; Abcam, United States), anti-PSD95 (1:500, mouse IgG; Abcam, United States). The following day, the cells and tissue sections were treated with goat anti-mouse IgG H&L (1:2000, Alexa fluor 647), goat anti-rabbit IgG H&L (1:2000, Alexa fluor 488) at room temperature for 2 h and the nuclei were counterstained for 10 min with DAPI. Immunoreactivity was visualized using a fluorescence microscope (AXIO Vert. A1&Imager A2, Carl Zeiss Microscopy GmbH, Germany). ImageJ software (NIH, Bethesda, MD) was used to randomly select five fields under the microscope and compare five views, The number of staining positive cells in the field was calculated by taking the mean of five fields to calculate the cell density. At the same time, the average fluorescence intensity of PSD95 in brain tissue was measured by Image J software (NIH, Bethesda, MD).