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Ab3697

Manufactured by Abcam
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Sourced in United Kingdom, United States

Ab3697 is a monoclonal antibody that recognizes the human Transferrin Receptor (CD71) protein. It can be used for the detection of this protein in various applications such as immunohistochemistry and flow cytometry.

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Lab products found in correlation

Ab14389, by Abcam (2 mentions) Ab92552, by Abcam (2 mentions) Ab36861, by Abcam (2 mentions) Lsm 510 meta, by Zeiss (1 mentions) Abs933, by Absin (1 mentions) Nitrocellulose membrane, by Solarbio (1 mentions) Ecl reagent, by Solarbio (1 mentions) Ab1031, by Merck Group (1 mentions) Bca kit, by Abcam (1 mentions) Ripa buffer, by Solarbio (1 mentions) Ab186838, by Abcam (1 mentions) Ab227387, by Abcam (1 mentions) Ab205718, by Abcam (1 mentions) Chip assay, by Merck Group (1 mentions) 4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (1 mentions) Fv1200 laser scanning confocal microscope, by Olympus (1 mentions) Goat anti rabbit igg hnl alexa fluor 488 ab150077, by Abcam (1 mentions) Bca protein assay kit, by Promega (1 mentions) Sc 52658, by Santa Cruz Biotechnology (1 mentions) Goat anti rabbit hrp, by Jackson ImmunoResearch (1 mentions)

23 protocols using ab3697

1

Quantification of Stem Cell Markers in Rat Tissues

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The colon, liver, spleen, kidney and lung tissues of the rats (n = 5) were fixed in 4% paraformaldehyde overnight and then dehydrated in 30% sucrose solution. The tissues were embedded in optimal cutting temperature (OCT) compound and cut into 5-μm-thick frozen sections. After soaking with PBS, the sections were placed in 5% normal goat serum (abs933, Absin, Shanghai, China) and incubated with anti-Bmi1 (1:200, ab14389, Abcam), anti-Musashi1 (1:200, c-135,721, Santa Cruz Biotechnology), anti-Sox9 (1:100, ab3697, Abcam) and anti-PCNA antibodies (1:200, ab92552, Abcam) at 4 °C overnight. Next, the sections were washed in PBS and incubated at 37 °C for 1 h with anti-mouse IgG (1:500, 8890, Cell Signaling Technology) and anti-rabbit IgG (1:500, 8889, Cell Signaling Technology). Sections were then stained with DAPI and anti-fading medium before observation by a laser scanning confocal microscope (LSM 510 META; Zeiss, Germany), and the results were semi-quantitatively analysed with ImageJ (National Institutes of Health, Bethesda, USA).
Li N., Zhang Y., Nepal N., Li G., Yang N., Chen H., Lin Q., Ji X., Zhang S, & Jin S. (2021). Dental pulp stem cells overexpressing hepatocyte growth factor facilitate the repair of DSS-induced ulcerative colitis. Stem Cell Research & Therapy, 12, 30.
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2

Western Blot Analysis of Chondrocyte Markers

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After lysing in RIPA buffer (Solarbio), cell lysates were centrifuged for protein collection. The concentration of protein sample was tested using a BCA kit (Abcam). Twenty microgram samples were loaded on sodium dodecyl sulfate/polyacrylamide gel electrophoresis and then transferred to nitrocellulose membranes (Solarbio). Next, the membranes were blocked in 5% non-fat milk, and then interacted with primary antibodies anti-Sry-type high-mobility-group box 9 (SOX9) (ab3697, 1:1000 dilution, Abcam), anti-collagen type II α 1 (COL2A1) (AB761, 1:100 dilution, Sigma–Aldrich), anti-Aggrecan (AB1031, 1:1000 dilution, Sigma–Aldrich), anti-TGFBR2 (ab186838, 1:500 dilution, Abcam) or anti-β-actin (ab227387, 1:5000 dilution, Abcam) and the secondary antibody conjugated by horseradish peroxidase (ab205718, 1:20000 dilution, Abcam). β-actin functioned as a loading control. Subsequently, the protein blots were developed via exposing to ECL reagent (Solarbio) and tested by ImageJ software (NIH, Bethesda, MD, U.S.A.). Relative protein level was normalized to the control group.
Zhang Q., Qiao X, & Xia W. (2020). CircSERPINE2 weakens IL-1β-caused apoptosis and extracellular matrix degradation of chondrocytes by regulating miR-495/TGFBR2 axis. Bioscience Reports, 40(11), BSR20201601.
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3

ChIP-PCR Analysis of Sox9 and Agc1 Enhancers

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We tested a defined, specific 48 bp sequence in the Sox9-binding enhancer region of collagen II α1 (COL2A1) for cartilage expression (39 (link)). We also assessed a defined, functional SOX9/HIF1a complex binding site in the 5'-UTR of AGC1 (40 (link)). To do so, we used primers corresponding to nucleotides 60–82 [forward] and 220–240 [reverse] in the AGC1 −640 to −510 promoter region (GenBank: AF031586). ChIP assay (Millipore) was carried out per manufacturer instructions, using Protein A agarose/Salmon Sperm DNA (50% slurry) reduce nonspecific background, and immunoprecipitation with SOX9 antibody (ab3697, Abcam). Following elution of histone/DNA complexes, and DNA recovery by phenol/chloroform extraction, DNA was resuspended in 10 µL TE buffer. PCR was conducted with 1 µL of eluate, 2 µL of 20 µM primers for Col2a1 and AGCN1 (Supplemental Table 1), 10 µL KAPA2G Fast ReadyMix PCR (Kapa Biosystems reagent from Sigma-Aldrich) and H2O up to 20 µL reaction volume.
Serrano R.L., Chen L.Y., Lotz M.K., Liu-Bryan R, & Terkeltaub R. (2018). Proteasomal function is impaired in human osteoarthritic chondrocytes and promotes decreased SOX9 and aggrecan levels. Arthritis & rheumatology (Hoboken, N.J.), 70(7), 1030-1041.
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4

SOX9 Immunofluorescence Assay for Micropatterned Cells

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Cells that have been grown on micropattern for 7, 14, and 21 days were fixed with methanol-DMEM series (50%, 70%, 80%, 90%, 100%) at −20 °C followed by washing three times in PBS at room temperature. After fixation, cells were permeabilized in PBS-T (0.05% Tween 20 in PBS) for 20 min at room temperature. Cells were blocked from unspecific binding antibody with 3% Bovine Serum Albumin (BSA, in PBST) for 60 min. The primary antibody against SOX9 (ab3697, Abcam) was then added to the samples and incubated 4 °C overnight. The samples were washed with PBS three times and incubated in the secondary antibody (1:200 goat anti-rabbit IgG HNL Alexa Fluor 488 ab150077, Abcam) in a dark chamber for 120 min and counterstaining with 4′,6-diamidino-2-phenylindole (DAPI, Thermo Fisher) for 10 min followed by washing three times with PBS. The stained cells were observed with a confocal microscope (Olympus Fv 1200 confocal laser scanning microscope).
Barlian A., Saputri D.H., Hernando A., Khoirinaya C., Prajatelistia E, & Tanoto H. (2022). Spidroin striped micropattern promotes chondrogenic differentiation of human Wharton’s jelly mesenchymal stem cells. Scientific Reports, 12, 4837.
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5

Quantifying Chondrocyte-MSC Interactions

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Protein expression levels of type II collagen (SC-52658, Santa Cruz, USA), SOX9 (ab3697, Abcam, USA), and GAPDH in co-cultured chondrocytes and MSCs were measured using Western blot. Cytosolic proteins were directly extracted with radio immune precipitation assay (RIPA) lysis buffer combined with a cocktail of protease and phosphatase inhibitors. The protein concentration was calculated using the BCA Protein Assay Kit (Promega). Western blot was performed using a kit (Beyotime, Biotechnology) according to the manufacturer's instructions, and ECL reagent was used to generate chemiluminescent signals.
Wang B., Liu W., Li J.J., Chai S., Xing D., Yu H., Zhang Y., Yan W., Xu Z., Zhao B., Du Y, & Jiang Q. (2021). A low dose cell therapy system for treating osteoarthritis: In vivo study and in vitro mechanistic investigations. Bioactive Materials, 7, 478-490.
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6

Immunohistochemical Staining of TBX18, Sox9, and α-SMA

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The TBX18-2 polyclonal antibody was generated under contract with Abgent, Inc (San Diego, CA) by attaching the peptide sequence (qqqlqkkrrklateea) to KLH and immunizing rabbits. Adult tissues were preserved in 4% paraformaldehyde in PBS. Post-fixed tissues were dehydrated, embedded in paraffin and sectioned for staining. Thin-sectioned tissues were stained with Hematoxylin and Eosin as previously described. Embryos were fixed in 4% PFA overnight at 4°C. They were then dehydrated and embedded in paraffin for sectioning. Four micron sections were deparaffinized, boiled for 30 minutes in citrate target retrieval solution pH 6.0, then treated with 3% H2O2 for thirty minutes. Slides were blocked with goat blocking serum for 1 hour, and then the TBX18-2 (1:800), Sox9 (1:400)(Abcam ab3697) and alpha-Smooth Muscle Actin (1:1000)(Abcam ab5694) antibodies were incubated overnight at 4°C. Sections were washed and a goat-anti-rabbit-HRP (Jackson Immunoresearch) secondary antibody was applied at 1:400 for one hour at room temperature. Sections were then stained with Perkin-Elmer TSA-Rhodamine for three minutes, washed and counterstained with Hoechst 33342.
Bolt C.C., Elso C.M., Lu X., Pan F., Kispert A, & Stubbs L. (2014). A distant downstream enhancer directs essential expression of Tbx18 in urogenital tissues. Developmental biology, 392(2), 483-493.
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7

ChIP Assay for SOX9 Transcriptional Regulation

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ChIP assays were performed using the Pierce Magnetic ChIP Kit according to the manufacturer’s instructions70 (link). Briefly, cross-linking with 1% formaldehyde was carried out in RTECs or renal tissues, followed by quenching with glycine, cell harvesting, and DNA fragmentation by sonication. Lysates were precleared for 1 h with Protein A + G magnetic beads (EMD Millipore). Precleared lysates were then incubated with 5 μg of anti-SOX9 antibodies (Abcam, ab3697) overnight at 4 °C, followed by addition of Protein A + G magnetic beads and incubation for 4 h at 4 °C. Subsequently, the beads were repeatedly washed, followed by elution of the protein–DNA complexes, reversal of cross-links, and DNA purification. Standard qPCR analysis was then carried out using primers spanning the promoters of target genes. The sequences of primers are shown in Supplementary Table 2.
Kim J.Y., Bai Y., Jayne L.A., Hector R.D., Persaud A.K., Ong S.S., Rojesh S., Raj R., Feng M.J., Chung S., Cianciolo R.E., Christman J.W., Campbell M.J., Gardner D.S., Baker S.D., Sparreboom A., Govindarajan R., Singh H., Chen T., Poi M., Susztak K., Cobb S.R, & Pabla N.S. (2020). A kinome-wide screen identifies a CDKL5-SOX9 regulatory axis in epithelial cell death and kidney injury. Nature Communications, 11, 1924.
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8

Mouse Embryo Histological and Molecular Analysis

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Mouse embryos were harvested from timed pregnant females and fixed in 4% paraformaldehyde solution at 4 °C overnight. Following dehydration through gradient ethanol, samples were embedded in paraffin and coronally sectioned at 8 μm. Slides were subjected to either hematoxylin/eosin staining for histological analysis or to in situ hybridization, as described previously (St Amand et al. 2000 (link)). For whole mount in situ hybridization, samples were dehydrated through gradient methanol after overnight fixation in 4% PFA and were subjected to in situ hybridization assay as described (Zhang et al. 1999 (link)). Antibodies used for immunohistochemistry staining include anti-Msx1 (R&D Systems, AF5045; 1:100), anti-Sox9 (Abcam, ab3697; 1:200), anti-Phospho-Smad1 (Ser463/465)/Smad5(Ser463/465)/Smad8(Ser426/428) (Millipore, AB3848; 1:100), anti-Ki67 (M3060; 1:200), anti-Sp7/Osterix (Abcam, ab22552; 1:100), anti-histone H3 (phosphor S10) (PHH3; Abcam, ab47297; 1:1000), anti-Caspase3 (Bioss, bs-0081R; 1:100) and anti-phospho-p38 MAPK (Thr180Tyr182) (Cell Signaling Technology, 4511s; 1:200).
Chen Y., Wang Z., Chen Y, & Zhang Y. (2018). Conditional deletion of Bmp2 in cranial neural crest cells recapitulates Pierre Robin sequence in mice. Cell and tissue research, 376(2), 199-210.
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9

Histological and Immunohistochemical Analysis

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Histology was performed using tissue fixed in 10% formalin for 24 h, dehydrated, and embedded in paraffin. Sections (7 μm) were cut and stained using hematoxylin and eosin (American Master Tech Scientific). Sections were also incubated with Bouin´s fluid overnight, counterstain with hematoxylin (Sigma), and then stained with Masson-Trichrome stain (American Master Tech Scientific). Immunohistochemistry was performed by staining tissue sections with antibodies against PCNA (biotinylated from Thermofisher MS-106-B; RRID:AB_64272), SOX9 (Abcam ab3697; RRID:AB_304012), glutamine synthetase (Abcam ab73593; RRID:AB_2247588), cytokeratin 19 (Abcam ab15463; RRID:AB_2281021), or phospho-p44/42 MAPK (Thr202/Tyr204) (Cell Signaling Technology #9101). Streptavidin-conjugated horseradish peroxidase (Biogenex) and the substrate 3,3′-diaminobenzidene (Vector Laboratories) were used followed by brief counterstaining with Mayer’s hematoxylin (Sigma).
Manieri E., Folgueira C., Rodríguez M.E., Leiva-Vega L., Esteban-Lafuente L., Chen C., Cubero F.J., Barrett T., Cavanagh-Kyros J., Seruggia D., Rosell A., Sanchez-Cabo F., Gómez M.J., Monte M.J., G. Marin J.J., Davis R.J., Mora A, & Sabio G. (2020). JNK-mediated disruption of bile acid homeostasis promotes intrahepatic cholangiocarcinoma. Proceedings of the National Academy of Sciences of the United States of America, 117(28), 16492-16499.
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10

Immunofluorescence Assay for HDAC9 and SOX9

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Breast cancer cells were fixed in 4% formaldehyde, permeabilized with 1% Triton X-100 and incubated with 1% bovine serum albumin for 3h to reduce non-specific binding at room temperature. The cells were then incubated with antibodies specific for HDAC9 (Abcam ab18970) or SOX9 (Abcam ab3697). Detection was performed using an Alexa-conjugated secondary antibody (Life Technologies). After washing, sections were counterstained with Hoechst (Sigma Aldrich) and mounted for fluorescence microscopy. Negative controls using rabbit or mouse IgGs were performed and no staining was observed in these conditions.
Lapierre M., Linares A., Dalvai M., Duraffourd C., Bonnet S., Boulahtouf A., Rodriguez C., Jalaguier S., Assou S., Orsetti B., Balaguer P., Maudelonde T., Blache P., Bystricky K., Boulle N, & Cavaillès V. (2016). Histone deacetylase 9 regulates breast cancer cell proliferation and the response to histone deacetylase inhibitors. Oncotarget, 7(15), 19693-19708.
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