Recombinant Anti-VEGF Receptor 1 antibody [Y103] (ab32152)

Blocking/Diluting buffer and concentration: 5% NFDM/TBST.

Immunohistochemical analysis of paraffin-embedded Human gastric carcinoma tissue labeling VEGF with ab32152, followed by a ready to use Goat Anti-Rabbit IgG H&L (HRP). Cytoplasmic staining on human gastric carcinoma. Counterstained with Hematoxylin. Heat mediated antigen retrieval using ab93684 (Tris/EDTA buffer, pH 9.0).

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is a ready to use Goat Anti-Rabbit IgG H&L (HRP).

VEGF Receptor 1 was immunoprecipitated from 0.35 mg mouse brain lysate 10μg with ab32152 at 1:30 dilution (2μg in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab32152 1:1000 dilution (2 μg/ml). VeriBlot for IP Detection Reagent (HRP) (ab131366) was used at 1:1000 dilution.

Lane 1: Mouse brain lysate 10μg.

Lane 2: ab32152 IP in mouse brain lysate.

Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab32152 in mouse brain lysate.

Blocking and dilution buffer and concentration: 5% NFDM/TBST.

Exposure time: 1 second.

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human abdominal aortic aneurysm (AAA) wall tissue sections labeling VEGF Receptor 1 with ab32152 at 1/100 dilution.

Resected aortic tissues were immersed in 10% neutral buffered formalin for at least 24 h for immunohistochemical staining. Tissue sample was embedded in paraffin; 4 µm sections were cut and mounted onto MAS-coated slides. The sections were deparaffinized, dehydrated, and boiled in a pressure cooker in 0.01 M citric acid buffer (pH 6.0) for 20 min. The sections were washed with phosphate-buffered saline and incubated with 3% H₂O₂ in absolute methanol for 5 min to inhibit any endogenous peroxidase activity. Sections were preincubated with 3% normal goat serum for 20 min to minimize nonspecific binding to VEGF Receptor 1, and incubated with ab32152 at 4°C overnight in a moist chamber. The section was washed with phosphate-buffered saline and then incubated with the appropriate secondary antibody for 30 min at room temperature. Staining was visualized with Vector DAB, and tissue section was then counterstained with hematoxylin.