Af4117

Manufactured by R&D Systems

AF4117 is a cell culture medium supplement produced by R&D Systems. It is designed to support the growth and maintenance of various cell types in vitro.

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Lab products found in correlation

5 protocols using af4117

Immunohistochemical Analysis of Vascular Markers

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Tissue sections were de-paraffined using xylene and a graded series of alcohols, non-specific background staining of endogenous peroxidase was treated with 1% hydrogen peroxide for 30 minutes, and sections were blocked with 1% bovine serum albumin prior to incubation with primary antibodies overnight at 4°C. Primary antibodies were directed against the endothelial marker CD31 (ab28364, Abcam; 1:50 dilution), the smooth muscle marker α-actin (ab5694, Abcam; 1:100 dilution), the progenitor marker CD34 (AF4117, R&D; 1:100 dilution), the endothelial marker VEGFR2 (ab2349, Abcam; 1:50 dilution), the M2 macrophage marker anti-transglutaminase 2 (TGM2; #37557; Cell Signaling; 1:50 dilution), proliferating cell nuclear antigen (PCNA; M0879, Dako ; 1:100 dilution) or the apoptosis marker cleaved caspase-3 (#9661; Cell Signaling; 1:100 dilution). Detection was performed using Dako EnVision^™ + Dual Link System-HRP (Dako; Carpinteria, CA), and counterstained with Mayer’s Hematoxylin. The integrated optical density of smooth muscle cells (SMC) in the neointima were analyzed using Image-Pro Plus 6.0 software (Media Cybernetics; Rockville, MD). Cells staining positively for CD34, VEGFR2, PCNA (proliferation index), or cleaved caspase-3 (apoptosis index) were directly counted in 4 high power fields, and compared to the total number of cells in the field.

Bai H., Kuwahara G., Wang M., Brownson K., Foster T., Yamamoto K., Xing Y, & Dardik A. (2014). Pretreatment of pericardial patches with antibiotics does not alter patch healing in vivo. Journal of vascular surgery, 63(4), 1063-1073.

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Immunohistochemical Analysis of Angiogenesis and Macrophage Markers

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Tissue sections were de-paraffined and blocked with 5% bovine serum albumin (BSA) prior to incubation with antibodies directed against CD34 (AF4117, R&D; 1:100 dilution), VEGFR2 (ab2349, Abcam; 1:50 dilution), CD68 [ED1] (ab31630, Abcam; 1:100 dilution), the M1 macrophage marker anti-inducible nitric oxide synthase (iNOS; #2977; Cell Signaling; 1:50 dilution), the M2 macrophage marker anti-TGM2 (#37557; Cell Signaling; 1:50 dilution), or the M2 macrophage marker anti-IL-10 (ab9969; Abcam; 1:50 dilution) overnight at 4°C. Secondary antibodies were: donkey anti-goat Alexa-Fluor-488, donkey anti-rabbit Alexa-Fluor-568, and chicken anti-mouse Alexa-Fluor-488 conjugated antibodies (Invitrogen; 1:1000 dilution). Sections were stained with 4’,6-diamidino-2-phenylindole (DAPI; Invitrogen) to mark cellular nuclei. Positively staining cells were counted in the presence of well-defined nuclei under 630x magnification in 5 high power fields, with the percentage of positive cells compared to the total number of cells in the field.

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Quantifying CD34+ Cells in Rat Brain

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Coronal sections of brain tissue (10 μm) were prepared using a freezing microtome (HS-4080, Hisure Scientific, Jinhua City, Zhejiang, China). The sections were then incubated overnight at 4°C with a primary antibody for the detection of CD34 (1:1000, AF-4117, R&D), followed by incubation for 2 hours at room temperature with a fluorescein-conjugated anti-IgG secondary detection antibody (1:100, TI-5000, Vector, Newark, CA). Immunofluorescence was then evaluated using an Olympus CKX5 microscope. For each rat, 5 sections of the right brain’s white matter were evaluated. The cells were counted in 3 areas of each section using automated Image J.

Zhu L., Yuan Q., Jing C., Sun L, & Jiang L. (2024). Angiogenic responses are enhanced by recombinant human erythropoietin in a model of periventricular white matter damage of neonatal rats through EPOR-ERK1 signaling. Journal of Neuropathology and Experimental Neurology, 83(3), 161-167.

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Comprehensive Immunohistochemistry and Immunofluorescence Protocol

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Primary antibodies included: anti-α-actin (Abcam, ab5694; IHC and IF, 1:100; WB, 1:1000); Dako actin (Smooth Muscle) Clone 1A4; anti-cleaved Caspase-3 (Cell Signaling #9661; IHC, 1:50; WB, 1:1000); anti-CD31 (Abcam, ab28364; IHC and IF, 1:50); anti-CD34 (R&D, AF4117; IF, 1:100; WB, 1:1000); anti-CD68 (ED1; Abcam, ab31630; IHC, 1:200; WB, 1:1000); anti-Eph-B4 (Santa Cruz, sc-5536; IF, 1:50); anti-Ephrin-B2 (Novus, NBP1-48610; IHC, 1:50); anti-GAPDH (Cell Signaling, 14C10; WB, 1:2000); anti-IL-10 (Abcam, ab9969; IF, 1:100); anti-Ki67 (Abcam, ab15580; IHC and IF, 1:100; WB, 1:1000); anti-phospho-mTOR (Cell Signaling, #2971; IHC, 1,50; WB,1:1000); anti-mTOR (Cell Signaling, #2972; WB, 1:1000); anti-transglutaminase 2 (TGM2; #37557; IF, 1:100); anti-VEGFR2 (ABCAM, ab2349; IF, 1:100; WB,1:1000); Secondary antibodies used for IF were: donkey anti-goat Alexa-Fluor-488, donkey anti-rabbit Alexa-Fluor-488, donkey anti-rabbit Alexa-Fluor-568, donkey anti-mouse Alexa-Fluor-568 and chicken anti-mouse Alexa-Fluor-488 conjugated antibodies from Invitrogen (1:1000). For IHC, sections were incubated with EnVision reagents for 1 h at room temperature and treated with Dako Liquid DAB + Substrate Chromogen System (Dako). Finally, the sections were counterstained with Mayer’s hematoxylin.

Bai H., Lee J.S., Chen E., Wang M., Xing Y., Fahmy T.M, & Dardik A. (2017). Covalent modification of pericardial patches for sustained rapamycin delivery inhibits venous neointimal hyperplasia. Scientific Reports, 7, 40142.

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Western Blot Analysis of Brain Proteins

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After protein extraction from brain tissues, proteases and phosphatases were inhibited by the addition of a Complete Mini Protease Inhibitor Cocktail Tablet (Roche, Basel, Switzerland) and the phosphatase inhibitor phenylmethylsulfonyl fluoride (10 mM). The protein concentrations of the samples were assessed using the Bradford protein assay. Total proteins (50 µg) were heated at 90°C for 10 minutes and then separated using 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The proteins were electrophoretically transferred onto nitrocellulose membranes, which were blocked using 5% skim milk. The membranes were then incubated with primary polyclonal antibodies recognizing EPOR (1:1000, FNab02816, Wuhan Fine Biotech, Wuhan, China), p-ERK1 (1:500, ab131438, Abcam, Cambridge, United Kingdom), ERK (1:1000, ab115799, Abcam), and CD34 (1:1000, AF-4117, R&D Systems, Minneapolis, MN) at 4°C overnight. Secondary antibodies (1:5000, ab6721 [Abcam] or 1:5000, sc-2352 [Santa Cruz, Dallas, TX]) were then reacted with the bound primary antibodies and visualized using enhanced chemiluminescence reagents (Pierce, Rockford, IL). The loading control comprised glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Image-J software was used for densitometry analysis.

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