Rabbit anti cd34 antibody

The Lucifer yellow-stained RVLM neurons that responded to EPO were stained for EPOR. To examine the presence of EPO and HIF-2α in the RVLM, we performed immunofluorescence staining before EPO superfusion. The following primary antibodies (1:400 dilution) were used for immunofluorescence: goat anti-erythropoietin antibody (Funakoshi, Tokyo, Japan), mouse anti-HIF-2α antibody (Abcam), rabbit anti-EPOR antibody (Sigma), and rabbit anti-CD34 antibody (Abcam). To confirm that the examined area was a C1 area, the existence of tyrosine hydroxylase (TH)-positive neurons in the RVLM was also examined using mouse anti-TH antibody (Sigma). The secondary antibodies for fluorescence staining (1:1,000 dilution) were Alexa Fluor 546 goat anti-rabbit IgG (Molecular Probes/Invitrogen, Eugene, OR) Alexa Fluor 633 donkey anti-goat IgG (Molecular Probes/Invitrogen), and Alexa Fluor 633 goat anti-mouse IgG (Molecular Probes/Invitrogen).

The isolation and identification of lung TCs were described by previous study 15. Briefly, mice were anaesthetized, of whom the lung tissues were isolated and digested by digestive fluid. After neutralizing digestive fluid, the fluid was filtered by 40 μm cell strainer and then centrifuged at 400 g for 5 min. Cells were collected and cultured in culture bottles in incubator (37°C, 5% CO₂). The supernatant was moved into new culture bottles 30 min. after cell adhesion on the plate. Cells were selected, purified and further amplified to meet the need for the experiment. The TCs were identified according to the morphology and immunofluorescent staining using mouse anti‐cKit antibody, rat anti‐vimentin antibody and rabbit anti‐CD34 antibody (1:200 dilution; Abcam, Cambridge, UK). After incubated overnight at 4°C with the first antibodies diluted in 1% bovine serum albumin (BSA) in PBS, the slides were washing in PBS for three times. Then, sections were incubated with PE conjugated antimouse secondary antibodies, PE conjugated anti‐rat secondary antibodies or FITC conjugated anti‐rabbit secondary antibodies according to the manufacturer (1:150 dilution; Abcam). DAPI was used to mark nuclear according to the manufacture (Abcam).

Ovaries from treated and control animals were fixed, dehydrated, vitrified, and embedded in paraffin. The ovarian sections (5 μm thick) were deparaffinized with xylene and hydrated using an ethanol gradient. The hydrated sections were washed in 3% hydrogen peroxide in methanol for 20 min at room temperature and blocked in PBS (Sigma) containing 3% BSA (Sigma) for 30 min. Sections were treated with primary antibodies including rabbit anti-CD34 antibody (1 : 200; Abcam, Cambridge, MA, USA), rabbit anti-VEGFA antibody (1 : 50; Abcam), rabbit anti-VEGFR1 antibody (1 : 200; Cell Signaling Technology, Danvers, MA, USA), or rabbit anti-VEGFR2 antibody (1 : 200; Abcam) for antibody detection at 4°C overnight. After washing, slides were then incubated with HRP labeled anti-rabbit antibody (Peroxidase reaction kits, Vector Laboratories, Burlingame, CA, USA). Peroxidase substrate was developed by using a DAB (3,39-diaminobenzidine) substrate kit (Vector Laboratories). Slides were counterstained with hematoxylin QS (Vector Laboratories) and were dehydrated and mounted with VectaMount Permanent Mounting Medium (Vector Laboratories).