Af1042

Manufactured by R&D Systems

Sourced in United States

AF1042 is a protein product manufactured by R&D Systems. It is a recombinant human Activin A protein. Activin A is a member of the transforming growth factor beta (TGF-β) superfamily and plays a role in regulating cell growth and differentiation.

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AF1042-SP (2 citations)

7 protocols using af1042

Immunoprecipitation of PDGF-BB Receptor

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Co-IP was performed using the Dynabeads Co-Immunoprecipitation Kit (ThermoFisher Scientific, Waltham, MA, USA), using polyclonal goat anti-mouse PDGF-BB antibody (Novus NBP1-52533) for immunoprecipitation. Briefly, a whole brain from a P21 WT (c57BL/6, n = 1) male mouse was harvested and homogenized in immunoprecipitation buffer, following a thoracotomy and PBS perfusion. Dynabeads were coupled with 10 ug of antibody per mg of beads. Whole-brain lysate (1.5 g protein) was processed with 1.5 g coupled beads according to the manufacturer’s instruction, and using the following extraction buffer: 100 mM NaCl, EDTA-free HALT Protease\Phosphatase Inhibitor (ThermoFisher Scientific, Waltham, MA, USA), 2 mM MgCl₂, and 1 mM DTT. Aliquots were collected at each step for downstream analysis. Eluted protein was immunoblotted under denaturing conditions (see Western blotting protocol above), using 1:1000 polyclonal goat anti-mouse PDGFRβ (R&D, AF1042), and 1:2000 donkey anti-goat AlexaFluor488 (Jackson ImmunoResearch, West Grove, PA, USA, 705-545-147).

Payne L.B., Abdelazim H., Hoque M., Barnes A., Mironovova Z., Willi C.E., Darden J., Houk C., Sedovy M.W., Johnstone S.R, & Chappell J.C. (2023). A Soluble Platelet-Derived Growth Factor Receptor-β Originates via Pre-mRNA Splicing in the Healthy Brain and Is Upregulated during Hypoxia and Aging. Biomolecules, 13(4), 711.

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Brain Barrier Integrity Evaluation

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After terminal anaesthesia, mice were perfused transcardially with saline followed by ice-cold 4% paraformaldehyde. Brains and spleens were post-fixed for 24 h, cryoprotected in 10% sucrose/PBS and sectioned (25 μm diameter) on a sledge microtome. Detection of IgG leakage to the brain parenchyma by immunostaining (Vector, NBA-2000, horse anti-mouse biotynilated IgG 1:500) was used to assess BBB permeability on brain sections as described earlier58 (link). Immunofluorescence was performed on free-floating brain sections using combinations of rabbit anti-Iba1 (WAKO, 019-19741, 1:500), rat anti-CD45 (AbDSerotec, MCA1388, 1:250), mouse anti-GFAP (Sigma, G3893, 1:500), rabbit anti-Claudin-5 (Invitrogen, 34-1600, 1:500) and goat anti-PDGFRβ (R&D Systems, AF1042, 1:500) antibodies. Sections were incubated in a primary antibody cocktail overnight followed by adequate fluorochrome (Invitrogen, A21206, donkey anti-rabbit Alexa 488 1:500; Invitrogen, A21203, donkey-anti-mouse Alexa 594 1:500)-conjugated antibodies. Biotinylated tomato lectin (Sigma-Aldrich, L0651-1 mg, 1:100) was used to visualize blood vessels, followed by streptavidin Alexa 350 conjugate (Invitrogen, S11249, 1:200). Images were captured using a Zeiss Axiovert 200M microscope with Axiovision 4.8 software.

Szalay G., Martinecz B., Lénárt N., Környei Z., Orsolits B., Judák L., Császár E., Fekete R., West B.L., Katona G., Rózsa B, & Dénes Á. (2016). Microglia protect against brain injury and their selective elimination dysregulates neuronal network activity after stroke. Nature Communications, 7, 11499.

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Immunohistochemical Staining of Ocular Tissues

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Globes were enucleated and fixed by emersion (2% formaldehyde in phosphate buffered saline pH 7.5, overnight at 4°C). Fixed globes were then opened with a razor blade and processed through a sucrose gradient prior to embedding in OCT media (cryosections) or dehydrated and embedded in paraffin using an automated tissue processor (Leica). 7 μm cryosections or 5μm paraffin sections were then used for immunostaining using standard methods. Paraffin sections were dewaxed and subjected to heat induced antigen retrieval (10 mM TRIS, 1 mM EDTA, 0.05% Tween20, pH 9, autoclaved 121°C 30 min liquid cycle) prior to staining. For staining, all sections were first blocked and permeabilized (5% donkey serum, 2.5% BSA, 0.5% Triton X100 in TBS, pH 7.5, 1 hr at room temperature) and then incubated overnight with appropriate primary antibodies diluted in additional blocking buffer. Slides were then washed (6 × 5 minutes, 0.05% Tween 20 in TBS, pH 7.5) and incubated for an additional 1hr at room temperature with appropriate Alexafluor-conjugated secondary antibodies (Invitrogen, Carlsbad CA, USA). Primary antibodies used: Goat anti PODXL, 1:250 (R&D systems AF1556), Goat anti CD31, 1:250 (R&D systems AF3628), Chicken anti GFP 1:5000 (Abcam, AB13970), Goat anti PDGFRB 1:100 (R&D systems AF1042). Slides were mounted and imaged using a Nikon A1R confocal microscope.

Liu P., Lavine J.A., Fawzi A., Quaggin S.E, & Thomson B.R. (2022). Angiopoietin-1 is required for vortex vein and choriocapillaris development in mice. Arteriosclerosis, thrombosis, and vascular biology, 42(11), 1413-1427.

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Ischemic Brain Tissue Analysis

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Animals were euthanized 2 weeks after ischemic onset. Brains were quickly removed and frozen, and coronal sections of 20 μm thickness were prepared. Infarction volumes were quantified on Nissl-stained sections using the “indirect” morphometric method with Image J software. Immunohistochemistry was performed as described before (Esposito et al., 2013 (link)) using primary antibodies anti-Doublecortin (DCX) (1:100, #18723 Abcam) and anti- BrdU (1:200, B35130 Invitrogen) as a marker of neurogenesis, and anti-type IV Collagen (1,10, #1340–01 SouthernBiotech) for vascular remodeling and anti-Ki67 (1,500, #1667 Abcam), as proliferation marker, for detecting angiogenesis. GFAP (1,200, #130300 Invitrogen), as a marker of astrocytes, PDGFβ (1,200, #AF1042 R&D systems), as marker of pericytes, Iba1(1,200, #019–19,741 WAKO or Abcam 5,076), as marker of microglia, or NeuN (1,200, #MAB377 Millipore) as marker of neurons, were co-stained with BDNF (1,200, # ab46176 Abcam) or VEGF (1,50, #sc152 Santa Cruz). Three nonoverlapping areas (0.125 mm² per area) were chosen in the boundary zone of the ischemic core to analyze the peri-infarct area.

Esposito E., Licastro E., Cuomo O., Lo E.H., Hayakawa K, & Pignataro G. (2023). Postconditioning promotes recovery in the neurovascular unit after stroke. Frontiers in Cellular Neuroscience, 17, 1260389.

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Characterizing PDGF-BB Signaling Pathways

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PDGF-BB (220-BB, R&D Systems). Antibodies used were as follows: anti-NCK1 (1/200, ab168940, Abcam), anti-NCK2 (1/200, ab109239, Abcam), anti-PDGF-B (1/50, AB23914, Abcam), anti-PDGFRβ (1/100, AF1042, R&D Systems), anti-NG2 (1/200, AB5320, Millipore), anti-MYH11 (1/200, AB53219, Abcam), anti-DESMIN (1/200, AF3844, R&D Systems), anti-TER119 eFluor 450 (1/50, 48-5921-82, eBioscience-ThermoFisher Scientific), anti-fibronectin (1/300, F3648, Sigma-Aldrich), anti-laminin (1/300, AB11575, Abcam), anti-collagen IV (1/300, AB769, Millipore), anti-GFP (1/300, G10362, Life Technologies), anti-ERG1/2/3 (1/100, SC353, Santa Cruz), anti-phospho-histone 3 (1/100, 06-570, Millipore), anti-α-smooth muscle actin CY3 (1/200, α-SMA, C6198, Sigma), anti-PDGFRβ 751 (1/1000, #4549, Cell Signaling), anti-PDGFRβ 1009 (1/1000, #3124, Cell Signaling), anti-PDGFRβ 1021 (1/1000, #2227, Cell Signaling), anti-PDGFRβ (1/1000, #3169, Cell Signaling), anti-p44/42 MAP kinase (1/1000, phospho-ERK, #9106, Cell Signaling), anti-44/42 MAP kinase (1/1000, total ERK, #9102, Cell Signaling), anti-pAKT (1/1000, #4060, Cell Signaling), and anti-panAKT (1/1000, total AKT, #4685, Cell Signaling). Appropriate secondary antibodies were conjugated to horseradish peroxidase (Vector Laboratories) or fluorescently labeled (Life Technologies). IB4 was purchased from Life Technologies.

Dubrac A., Künzel S.E., Künzel S.H., Li J., Chandran R.R., Martin K., Greif D.M., Adams R.H, & Eichmann A. (2018). NCK-dependent pericyte migration promotes pathological neovascularization in ischemic retinopathy. Nature Communications, 9, 3463.

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Immunofluorescence Imaging of PDGF and PDGFR in 3D Cell Cultures

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IHCs were performed on paraffin sections (4–5 μm) of NMF and BJ3Z cells cultured in 3D colonies as described [30 (link)]. They used antibodies directed against PDGF-A (sc-7958; rabbit polyclonal, Santa Cruz), PDGF-B (sc-7878; rabbit polyclonal, Santa Cruz), PDGFR-α (AF1062; goat polyclonal, R&D systems) and PDGFR-β (AF1042; goat polyclonal, R&D systems). Briefly, sections were deparaffinized and antigen retrieval was performed in a pressure cooker (Bio-care Medical) at 20 psi for 5 min in citrate buffer (10 mM sodium citrate, 0.05% Tween-20, pH 6.0). Sections were blocked 30 min with 10% normal goat serum and primary antibodies were applied for 1 hr. Fluorescent secondary antibodies were: Alexa fluor 555 (red) donkey anti-goat IgG (1:300) and Alexa fluor 488 (green) goat anti- rabbit IgG (1:400; both Invitrogen). Cell nuclei were counterstained with 4-6-Diamidino-2-phenylindole (DAPI). Fluorescent images were obtained using a Nikon Eclipse E600 fluorescent microscope coupled to a RGB-MSC micro color camera, and Image Pro Plus software version 4.5 (Media Cybernetics, Silver Spring, MD).

Pinto M.P., Dye W.W., Jacobsen B.M, & Horwitz K.B. (2014). Malignant stroma increases luminal breast cancer cell proliferation and angiogenesis through platelet-derived growth factor signaling. BMC Cancer, 14, 735.

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Multiplex Immunofluorescence for Glia and Neuron Markers

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Ms-Olig2: Millipore MABN50, clone 211F1.1, 1:100 (RRID:AB_10807410). Rb-Olig2: Millipore AB9610, 1:500 (RRID:AB_570666). Rb-Ki67: ThermoFisher, PA5-19462, 1:200 (RRID:AB_10981523). Ms-CC1: Millipore Sigma, OP80, 1:100 (RRID:AB_2057371). Gt-PDGFRα: R&D Systems AF1062, 25 μg, 1:200; 100 μg, 1:60 (RRID:AB_2236897). Gt-PLP: Santa Cruz Biotechnology, G-17, sc-23570 (discontinued), 1:250 (RRID:AB_2165797). Rb-NF200: Sigma-Aldrich N4142, 1:250 (RRID:AB_477272). Ms-Nestin: Millipore Sigma, clone rat-401, MAB353, 1:500 (RRID:AB_94911). Gt-PDGFRβ: R&D Systems AF1042, 1:200 (RRID:AB_2162633). Rb-Laminin: Millipore AB2034, 1:100 (RRID:AB_91209). Rb-NG2: Millipore AB5320, 1:200 RRID:AB_91789). Rb-ALDH1A2: Abcam AB75674, 1:200 (RRID:AB_10676130). Rb-IBA1: Wako Chemicals 019-19741, 1:750 (RRID:AB_839504). Rb-GFAP: Millipore AB5804, 1:500 (RRID:AB_2109645). Rt-CTIP2: Abcam AB18465, 1:500 (RRID:AB_2064130). Rb-TBR1: Abcam AB31940, 1:50 (RRID:AB_2200219). Ms-SATB2: Abcam AB515102, 1:500 (RRID:AB_882455).

Morrison V.E., Smith V.N, & Huang J.K. (2020). Retinoic Acid Is Required for Oligodendrocyte Precursor Cell Production and Differentiation in the Postnatal Mouse Corpus Callosum. eNeuro, 7(1), ENEURO.0270-19.2019.

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