Total cellular protein was extracted using RIPA buffer (Sigma-Aldrich) and quantified with BCA assay (Sigma-Aldrich). SPC25 and PDGF were measured with ELISA kits from MyBiosource and R&D Biosystem, respectively. Western blot was performed with the following antibodies: a rabbit anti-human SPC25 (1:750; Ab236972, Abcam, Dallas, TX, USA), a mouse anti-human Egr-1 (1:2,000; Ab55160, Abcam), a rabbit anti-human PDGF (1:1,000; Ab23914, Abcam), and a mouse anti-human GAPDH antibody (1:1,000; Ab8245, Abcam). All secondary antibodies were from Jackson ImmunoResearch Labs (West Grove, PA, USA). Quantification was done with ImageJ (NIH, Bethesda, MA, USA). Immunocytochemistry was done with a mouse anti-human PDGFR alpha antibody (1:50; Ab96569, Abcam).
Ab96569
Manufactured by Abcam
Ab96569 is a laboratory equipment product. It is a device used for specific scientific purposes, but a detailed description cannot be provided while maintaining an unbiased and factual approach.
Automatically generated - may contain errors
Lab products found in correlation
Ab93876, by Abcam (2 mentions)
Ab19027, by Abcam (2 mentions)
Ab33734, by Abcam (2 mentions)
Ab189267, by Abcam (2 mentions)
Ab185716, by Abcam (2 mentions)
Ab34710, by Merck Group (2 mentions)
Ab11306, by Abcam (2 mentions)
Ab74524, by Abcam (2 mentions)
Ab75769, by Abcam (2 mentions)
Ab76956, by Abcam (2 mentions)
Ab108337, by Abcam (2 mentions)
Ab203887, by Abcam (2 mentions)
Ab107595, by Abcam (2 mentions)
Mab8887, by Abcam (2 mentions)
Ab109186, by Abcam (2 mentions)
Ab6319, by Abcam (2 mentions)
Ab155279, by Abcam (2 mentions)
Ab201980, by Abcam (2 mentions)
Ab11132, by Abcam (2 mentions)
Ab6326, by Abcam (2 mentions)
6 protocols using ab96569
1
Protein Quantification and Analysis
2
Characterization of Musculoskeletal Cell Markers
3
Characterization of Musculoskeletal Cell Markers
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Primary antibodies used were specific for Collagen type I (Abcam, ab34710, 1:100 dilution), Collagen type II (Millipore, MAB8887, 1:100 dilution), murine CD200 (Abcam, ab33734, 1:100 dilution), Nestin (Abcam, ab11306, 1:200 dilution), Gremlin 1 (Abcam, ab189267, 1:50 dilution), COMP (Abcam, ab74524, 1:50 dilution), Aggrecan (Abcam, ab3778, 1:100 dilution), Thy1.2 (Invitrogen, cat no. 14-0902-82, 1:50 dilution, 6C3 (Invitrogen, cat no. 14-5891-82 (1:50 dilution), CD105 (Abcam, ab107595, 1:100 dilution), Runx2 (Abcam, ab76956, 1:200 dilution), Alpl (Abcam, ab108337, 1:100 dilution), Osteocalcin (Abcam, ab93876, 1:100 dilution), CD146 (Abcam, ab75769, 1:100 dilution), CD140α (Abcam, ab96569, 1:100 dilution), CD200 (Abcam, ab203887, 1:200 dilution), Tartrate Resistant Acid Phosphatase (TRAP) (Abcam, ab185716, 1:50 dilution), and Cathepsin K (Abcam, ab19027, 1:200 dilution).
4
Immunohistochemical Analysis of Neural Cell Markers
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A group of sections were randomly selected from the three additional groups of sections from each mouse. The sections were rinsed with PBS-T, successively infiltrated with 2 mol/L HCl for 10 min (only required for the anti-BrdU antibody), subjected to antigen retrieval in citrate buffer (0.01 M, 99°C) for 30 min, and incubated with normal goat serum for 2 h at 37°C. Then, the primary anti-CNPase (ab6319, Abcam), anti-Olig2 (rabbit, ab109186, Abcam), anti-SOX10 (ab155279, Abcam), anti-bromodeoxyuridine (BrdU; ab6326, Abcam), anti-5-HT1AR (ab85615, Abcam), anti-platelet-derived growth factor alpha receptor (PDGFαR; ab96569, Abcam), anti-Aβ (ab11132, Abcam), and anti-CDKN2A/p16INK4a (p16; ab201980, Abcam) antibodies were added at a dilution of 1:500 in PBS, incubated at 4°C for 72 h and then rewarmed at 37°C for 2 h. The appropriate DyLight 405, DyLight 488, and DyLight 549-conjugated secondary antibodies (A23140, A23210, and A23320, respectively, Abbkine, P. R. China) were incubated with the sections at a 1:200 dilution. Finally, the sections were mounted on gelatin-coated slides with antifade solution to reduce fluorescence quenching.
5
Immunohistochemistry for Ki67 and PDGFRα
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Biopsies were fixed in formalin, dehydrated in 70% ethanol, and embedded in paraffin. Sections were cut (5 μm) and rehydrated. Heat-induced epitope retrieval (Reveal Decloaker 10x, Biocare Medical) was performed for 20 minutes, followed by 0.3% peroxidase ((HX0635-3 MilliporeSigma), and blocked in blocking buffer (MilliporeSigma) for 1 hour at room temperature. Specimens were incubated at 4°C overnight with mouse anti-Ki67 (1:400, ab156956, Abcam) or mouse anti-PDGFRα (16AI; 1:100, ab96569, Abcam), then washed, incubated for 30 minutes in a detection reagent (HRP-Polymer, Biocare Medical), and developed with DAB (Betazid DAB, Biocare Medical). Sections were counterstained with hematoxylin and visualized and photographed using an Olympus BX43 microscope (Olympus Life Science).
6
Comprehensive Immunolabeling of Mouse Brain Sections
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A group of sections was randomly selected from three additional groups of sections per mouse. The sections were rinsed in PBS-T 4 times 15 min each as previously described and then in ltrated in 2 mol/L HCl successively for 10 min at 0 °C, 25 °C and 37 °C. After the sections were repaired in boric acid solution for 10 min, nonspeci c binding sites were blocked by incubating the sections with normal goat serum for 2 hours at 37 °C. Then, the primary anti-CNPase (mouse, ab6319, Abcam), anti-Olig2 (rabbit, ab109186, Abcam), anti-SRY-related HMG-box 10 (SOX10; rabbit, ab155279, Abcam), anti-platelet-derived growth factor alpha (PDGFα; mouse, ab96569, Abcam), anti-bromodeoxyuridine (BrdU; rat, ab6326, Abcam), anti-Aβ (mouse, ab11132, Abcam) and anti-CDKN2A/p16INK4a (p16; mouse, ab201980, Abcam) antibodies were added at a dilution of 1:500 in PBS and incubated at 4 °C for 72 hours and then rewarmed at 37 °C for 2 hours. Then, secondary antibodies (1:200) were the appropriate DyLight 405, DyLight 488, and DyLight 549 (Abbkine)-conjugated antibodies. Finally, the sections were mounted on gelatin-coated slides with antifade solution to reduce uorescence quenching.
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