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Af534

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AF534 is a recombinant human Activin A protein. Activin A is a member of the transforming growth factor beta (TGF-β) superfamily of proteins and regulates a variety of cellular processes, including cell growth, differentiation, and apoptosis.

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Lab products found in correlation

Imagene red c12rg lacz gene expression kit, by Thermo Fisher Scientific (2 mentions) Dy531, by R&D Systems (2 mentions) Anti mouse f4 80, by Bio-Rad (1 mentions) Ab103791, by Abcam (1 mentions) Ab16894, by Abcam (1 mentions) Ab150064, by Abcam (1 mentions) Ab66705, by Abcam (1 mentions) Ab213676, by Abcam (1 mentions) Ab150109, by Abcam (1 mentions) Bond rx autostainer platform, by Leica camera (1 mentions) Horse anti goat igg, by Vector Laboratories (1 mentions)

7 protocols using af534

1

Fluorescent SA-β-gal Labeling and Immunofluorescence

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For the fluorescent SA-β-gal labeling, tissue slides were exposed to the C12RG substrate at 37 °C according to manufacturer’s instructions (ImaGene Red C12RG lacZ Gene Expression Kit, Molecular Probes, I2906)48 (link),49 . Subsequently, for immunofluorescence analysis, slides were fixed with 4% paraformaldehyde for 10 min at room temperature and regular immunofluorescence was performed following standard protocols and those previously described14 (link). The following antibodies were used: anti-mouse uPAR uPAR (AF534,R&D, DCL0521042; 1:50 dilution) and anti-mouse F4/80 (Bio-Rad, CI:A3-1, 155529; 1:100 dilution). For quantification, five high-power fields per section were counted and averaged to quantify the percentage of SA-β-gal+, uPAR+ and F4/80+ per DAPI-positive cells. For colocalization analysis, Pearson coefficient was calculated using ImageJ.
Amor C., Fernández-Maestre I., Chowdhury S., Ho Y.J., Nadella S., Graham C., Carrasco S.E., Nnuji-John E., Feucht J., Hinterleitner C., Barthet V.J., Boyer J.A., Mezzadra R., Wereski M.G., Tuveson D.A., Levine R.L., Jones L.W., Sadelain M, & Lowe S.W. (2024). Prophylactic and long-lasting efficacy of senolytic CAR T cells against age-related metabolic dysfunction. Nature Aging, 4(3), 336-349.
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2

Quantifying Mouse Serum suPAR Levels

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To evaluate suPAR concentration in mouse serum, a kit from R&D systems (DY531) that has been validated by the manufacturer for detection of mouse suPAR in cell supernatant was used. This assay uses polyclonal goat IgG mouse uPAR antibody (R&D systems; Cat# AF534) for suPAR detection. The assays were performed as per the manufacturer’s protocol. suPAR standard protein was serially diluted (concentrations ranging from 337pg/ml to 2500pg/ml) in the provided reagent diluent (0.05% Tween-20 in PBS) to generate standard curves. Standard curve derivation was repeated in serum from PLAUR −/− mice (gift from Dr. Nicholai Sidenius, Unit of Cell Matrix Signaling IFOM, the FIRC Institute for Molecular Oncology, Milan, Italy). Pooled serum from 6 wild type mice was used to estimate the basal concentration of suPAR in mice. All samples were assayed in duplicate. The mean co-efficient of variation in these assays was 5%.
Spinale J.M., Mariani L.H., Kapoor S., Zhang J., Weyant R., Song P.X., Wong H.N., Troost J.P., Gadegbeku C.A., Gipson D.S., Kretzler M., Nihalani D, & Holzman L.B. (2014). A reassessment of soluble urokinase-type plasminogen activator receptor in glomerular disease. Kidney international, 87(3), 564-574.
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3

Immunofluorescence Analysis of TLR2, Serpin E1, PLAUR, BNP, OSM, PGP9.5, NeuN, and PLAUR

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Paraffin sections were de‐paraffinized, rehydrated, permeabilized, followed by incubation with rabbit antibody to TLR2 (1:300, Abcam ab213676), Serpin E1 (1:300, Abcam ab66705), PLAUR (1:300, Abcam ab103791), BNP (1:100, Abcam ab236101), OSM (1:75, Thermo PA576861), mouse antibody to PGP9.5 (1:300, Abcam ab8189), NeuN (1:500, Novus NBP1‐92693), TLR2 (1:300, Abcam ab16894), or Antigen Affinity‐purified polyclonal goat IgG against PLAUR (10ug/ml, AF534, R&D) in blocking solution (4°C, overnight). The samples were washed in PBS and incubated with donkey anti‐rabbit Alexa 594 (1:500, Abcam ab150064) or anti‐mouse Alexa 488 (1:500, Abcam ab150109). Subsequent to a final wash, specimens were mounted onto slides using prolonged anti‐fade reagents containing DAPI (ThermoFisher Scientific) and captured using an IX73 Olympus microscope. Fluorescence intensity was analyzed using CellSens Dimension Imaging software and Image J.
Chen W., Li Y., Steinhoff M., Zhang W., Buddenkotte J., Buhl T., Zhu R., Yan X., Lu Z., Xiao S., Wang J, & Meng J. (2022). The PLAUR signaling promotes chronic pruritus. The FASEB Journal, 36(6), e22368.
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4

Quantifying Mouse Serum suPAR Levels

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To evaluate suPAR concentration in mouse serum, a kit from R&D systems (DY531) that has been validated by the manufacturer for detection of mouse suPAR in cell supernatant was used. This assay uses polyclonal goat IgG mouse uPAR antibody (R&D systems; Cat# AF534) for suPAR detection. The assays were performed as per the manufacturer’s protocol. suPAR standard protein was serially diluted (concentrations ranging from 337pg/ml to 2500pg/ml) in the provided reagent diluent (0.05% Tween-20 in PBS) to generate standard curves. Standard curve derivation was repeated in serum from PLAUR −/− mice (gift from Dr. Nicholai Sidenius, Unit of Cell Matrix Signaling IFOM, the FIRC Institute for Molecular Oncology, Milan, Italy). Pooled serum from 6 wild type mice was used to estimate the basal concentration of suPAR in mice. All samples were assayed in duplicate. The mean co-efficient of variation in these assays was 5%.
Spinale J.M., Mariani L.H., Kapoor S., Zhang J., Weyant R., Song P.X., Wong H.N., Troost J.P., Gadegbeku C.A., Gipson D.S., Kretzler M., Nihalani D, & Holzman L.B. (2014). A reassessment of soluble urokinase-type plasminogen activator receptor in glomerular disease. Kidney international, 87(3), 564-574.
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5

Senescence-Associated Beta-Galactosidase Staining

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SA-β-gal staining was performed as previously described76 (link) at pH 5.5 for mouse tissues. Specifically, fresh frozen tissue sections were fixed with 0.5% glutaraldehyde in phosphate-buffered saline (PBS) for 15 min, washed with PBS supplemented with 1 mM MgCl2 and stained for 5–8 h in PBS containing 1 mM MgCl2, 1 mg ml−1 X-gal, 5 mM potassium ferricyanide and 5 mM potassium ferrocyanide. Tissue sections were counterstained with eosin. Three fields per section were counted with ImageJ and averaged to quantify the percentage of SA-β-gal+ area per field. For the fluorescent SA-β-gal labelling, tissue slides were exposed to the C12RG substrate at 37°C according to manufacturer’s instructions (ImaGene Red C12RG lacZ Gene Expression Kit, Molecular Probes, I2906)77 (link),78 (link). Subsequently, for IF analysis, slides were fixed with 4% PFA for 10 minutes at room temperature and proceed with regular IF as performed following standard protocols and previously described27 (link). The following antibodies were used: anti-mouse uPAR (R&D, AF534, 1:100)
Eskiocak O., Chowdhury S., Shah V., Nnuji-John E., Chung C., Boyer J.A., Harris A.S., Habel J., Sadelain M., Beyaz S, & Amor C. (2024). Senolytic CAR T cells reverse aging-associated defects in intestinal regeneration and fitness. bioRxiv.
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6

Immunohistochemistry and Electron Microscopy of Mouse Kidney

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Immunostains for uPAR were performed on formalin-fixed paraffin-embedded (FFPE) mouse kidneys using standardized immunoperoxidase protocol with goat anti-mouse uPAR antibody (AF534, R&D System) following antigen retrieval at 100°C in pH 8.0, a secondary antibody Rabbit anti-goat HRP, and BOND Polymer detection RTU KIT with mouse HRP Polymer (Abcam) and DAB for detection. The IgG1 immunostain was performed on mouse FFPE kidneys with an anti-human IgG1 antibody (RevMAb Biosciences) using a standardized indirect immunofluorescence technology; human FFPE tonsil tissue was used as positive control. Immunostains against Integrin Beta 3 were performed on mouse frozen kidneys with an anti-Integrin Beta 3 (AP-5) antibody (Richard H. Aster, Blood Center of Wisconsin) using a standardized immunofluorescence protocol. All immunostains were preformed on a Leica Bond RX Autostainer platform. For electron microscopy, ultrathin (80 nm) sections of the glutaraldehyde-fixed, Epon-embedded mouse kidneys were stained with 2% uranyl acetate. Sections were examined in a Tecnai G212 transmission electron microscope at 80 kV, with images obtained with a Hammamatsu camera model Orca HR.
Harel E., Shoji J., Abraham V., Miller L., Laszik Z.G., King A., Dobi D., Szabo G., Hann B., Sarwal M.M., Craik C.S, & Vincenti F. (2020). Further Evidence that the Soluble Urokinase Plasminogen Activator Receptor Does Not Directly Injure Mice or Human Podocytes. Transplantation, 104(1), 54-60.
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7

Immunohistochemical and Senescence Analysis

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Tissues were fixed overnight in 10% formalin, embedded in paraffin and cut into 5-μm sections. Sections were subjected to hematoxylin and eosin (H&E) staining. Immunohistochemical staining was performed following standard protocols. The following antibodies were used: anti-mouse uPAR (R&D, AF534, lot DCL0521042, 1:50), Horse anti-goat IgG (Vector laboratories, 30116; lot ZH0526). Three fields per section were counted per sample with ImageJ and averaged to quantify the percentage of uPAR+ area per field. SA-β-gal staining was performed as previously described47 (link) at pH 5.5 for mouse tissues. Specifically, fresh frozen tissue sections were fixed with 0.5% glutaraldehyde in phosphate-buffered saline (PBS) for 15 min, washed with PBS supplemented with 1 mM MgCl2 and stained for 5–8 h in PBS containing 1 mM MgCl2, 1 mg ml−1 X-gal, 5 mM potassium ferricyanide and 5 mM potassium ferrocyanide. Tissue sections were counterstained with eosin. Three fields per section were counted with ImageJ and averaged to quantify the percentage of SA-β-gal+ area per field.
Amor C., Fernández-Maestre I., Chowdhury S., Ho Y.J., Nadella S., Graham C., Carrasco S.E., Nnuji-John E., Feucht J., Hinterleitner C., Barthet V.J., Boyer J.A., Mezzadra R., Wereski M.G., Tuveson D.A., Levine R.L., Jones L.W., Sadelain M, & Lowe S.W. (2023). Prophylactic and long-lasting efficacy of senolytic CAR T cells against age-related metabolic dysfunction. Research Square.
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