Rabbit anti plgf

Rabbit anti-actin (Sigma), rat anti-IL1-beta/IL-1F2 (R&D), rabbit anti-TGF-beta (Nordic Biosite), rat anti-GM-CSF (Abcam), rat anti-IL10 (Abcam), goat anti-M-CSF (Abcam), goat anti-VEGFD (Abcam), rabbit anti-VEGF (Abcam), rabbit anti-VEGFC (Abcam), rabbit anti-PlGF (Abcam), rabbit anti-IL-6 (Abcam), rabbit anti-phospho (Ser727) STAT3 (Cell Signaling), rabbit anti-phospho, (Tyr705) STAT3 (Cell Signaling), rabbit anti-STAT3 (Cell Signaling). Anti-GM-CSF, IL-10 and IL-1beta were monoclonal antibodies, all other antibodies were polyclonal.

The pretreated HUVECs were washed by pre-cooling PBS three times, and the proteins were extracted by RIPA lysis containing 1% PMSF (Solarbio, Beijing, China). The concentration of proteins was measured according to the BCA protein assay kit (Solarbio, Beijing, China). The proteins were boiled for 5 min in SDS-PAGE loading buffer, and 30 μg of proteins was loaded to each lane and separated by 12% SDS-PAGE gels and transferred onto polyvinylidene fluoride (PVDF; Millipore, Billerica, MA, USA) membranes. The membranes were blocked by 5% non-fat milk for 1 h and then immersed in primary antibodies overnight at 4 °C; the primary antibodies are as follows: rabbit anti-GAPDH (1:10000; Proteintech, Chicago, IN, USA), rabbit anti-PLGF (1:1000; Abcam, Cambridge, UK), rabbit anti-SCF (1:10000; Abcam), and rabbit anti-VEGFR2 (1:1000; Cell Signaling Technology, USA). The membranes were incubated with horseradish peroxidase-conjugated secondary antibodies (1:10000; Proteintech) for 1 h. Chemiluminescence reagents (Millipore) were used for the development of band visualization. ImageJ software was used to analyzed the protein expression levels.

Human decidual tissues were labeled with rabbit anti-PlGF (Abcam) for immunohistochemical staining as previously described (24 (link)). Specifically, the formalin-fixed decidual tissues were dehydrated in ethanol, embedded with paraffin, and cut into slices. After the deparaffinization and rehydration, the sections were pretreated with sodium citrate buffer (0.01M, pH=6.0) for antigen retrieval, followed by blocking the endogenous peroxidase activity with 3% H₂O₂ and 5% bovine serum albumin. Then, the sections were incubated with rabbit anti-PlGF antibodies (1:100, Abcam), or rabbit IgG overnight at 4°C. After washing with PBS, the sections were incubated with secondary antibody for 30min at room temperature, labeled with 3,3’-diaminobenzidine, and counterstained with hematoxylin.