Brains were taken out immediately after perfusion and were postfixed for 1 h (P0), 1–2 h (P4–P14) or 1–4 h (P21). After the postfixation, the brains were washed in 0.1 m phosphate buffer for 20 min and were sunk in 30% sucrose/0.1 m phosphate buffer. Sections were cut as described above for in situ hybridization. The following primary antibodies were used: β-galactosidase (β-gal; 1:100, goat, catalog #55976, Cappel; 1:500, chicken, catalog #ab9361, Abcam); SOX6 (1:100, rabbit, catalog #ab30455, Abcam); SP8 (1:100, goat, catalog #sc-104661, Santa Cruz Biotechnology); CTIP2 (1:200, rat, catalog #ab18465, Abcam); TBR1 (1:200, rabbit, catalog #ab31940, Abcam; 1:200, chicken, catalog #AB2261, Millipore); PV (1:500, rabbit, catalog #PV27, SWANT); somatostatin (SST; 1:100, rat, catalog #MAB354, Millipore); LIM Homeobox 6 (LHX6; 1:50, mouse, catalog #sc-271433, Santa Cruz Biotechnology); vesicle-associated membrane protein 2 (VAMP2; 1:200, rabbit, catalog #104 202, Synaptic Systems); NetrinG1 (1:100, goat, catalog #AF1166, R&D Systems); and cleaved caspase 3 (1:100, rabbit, catalog #D175, Cell Signaling Technology). Secondary antibodies conjugated with Cy2, Cy3, or Cy5 were obtained from Jackson ImmunoResearch.
Ab30455
Manufactured by Abcam
Sourced in United Kingdom, United States
Ab30455 is a lab equipment product. It is a device designed for use in scientific research and laboratory settings. The core function of this product is to provide a specific capability or measurement required for conducting experiments or analyses. No further details about the intended use or specific applications of this product can be provided in an unbiased and factual manner.
Automatically generated - may contain errors
Lab products found in correlation
Ab18465, by Abcam (2 mentions)
Ab39670, by Abcam (2 mentions)
Ab93855, by Abcam (2 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Ab9361, by Abcam (1 mentions)
Ab31940, by Abcam (1 mentions)
Sc 104661, by Santa Cruz Biotechnology (1 mentions)
Ab2261, by Merck Group (1 mentions)
Mab354, by Merck Group (1 mentions)
Sc 271433, by Santa Cruz Biotechnology (1 mentions)
Af1166, by R&D Systems (1 mentions)
Cyanine3 (cy3), by Jackson ImmunoResearch (1 mentions)
Ab231720, by Abcam (1 mentions)
Ab77232, by Abcam (1 mentions)
Ab91506, by Abcam (1 mentions)
Ab11083, by Abcam (1 mentions)
Horse serum, by Thermo Fisher Scientific (1 mentions)
Mirvana mirna inhibitor, by Thermo Fisher Scientific (1 mentions)
Lipofectamine rnaimax transfection reagent, by Thermo Fisher Scientific (1 mentions)
Dulbecco s modified eagle medium dmem, by Thermo Fisher Scientific (1 mentions)
10 protocols using ab30455
1
Immunohistochemical Analysis of Murine Brain Tissue
2
Myricetin Modulates Skeletal Muscle Metabolism
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Myricetin (DY0103, HPLC ≥98%) was purchased from MUST Biotechnology Co., Ltd. (Chengdu, China) for animal study. For cell experiments, Myricetin (70050) and DMSO (D2650) was obtained from Sigma-Aldrich (St. Louis, MO, USA). Dulbecco’s modified Eagle medium (DMEM) and horse serum (16050130) were bought from Gibco (Carlsbad, CA). Fetal bovine serum (FBS) was purchased from Hyclone Laboratories, Inc. (Logan, UT, USA). Cell Counting Kit-8 (CCK-8) was purchased from Dojindo (Kumamoto, Japan). mirVana™ miRNA Inhibitors (rno-miR-499-5p, MH11352), mirVanaTM miRNA mimics (rno-miR-499-5p, MC11352), Lipofectamine RNAiMAX Transfection Reagent (13778083) and antibody against PGC-1α (PA5–38021) were bought from Invitrogen (Massachusetts, USA). Antibodies against slow skeletal myosin heavy chain (ab11083), fast skeletal myosin heavy chain (ab91506), Sox6 (ab30455), tnni1 (ab231720) and myoglobin (ab77232) were purchased from Abcam (Cambridge, UK). Antibody against Cytochrome C (Cyt C, sc-13,561) and β-actin (sc-47778) were purchased from Santa Cruz Biotechnology (CA, USA).
3
Multiplexed Labeling of Brain Cells
4
Protein Expression Analysis via Western Blot
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The cells were dissolved in RIPA lysis buffer with 1% phenylmethylsulfonyl fluoride and centrifuged (12,000×g, 15 min) at 4 °C. Pierce™ BCA protein assay kit (Thermo Fisher Scientific, Rockford, USA) was used to detect the protein concentration, and the SDS-PAGE was used to separate the proteins. Afterwards, the proteins were transferred onto a PVDF membrane and blocked with 5% fat-free milk at room temperature for 1 h. Next, the membrane was incubated with primary antibodies (anti-SOX6, ab30455, Abcam, 1:1000; anti-GAPDH, ab8245, Abcam, 1:1000; anti-Bcl-2, ab196495, Abcam, 1:1000; anti-Bax, ab53154, Abcam, 1:1000; anti-Cleaved-caspase 3, #9661, CST, 1:1000) at 4 °C overnight and washed with TBST for three times (5 min/time) and then incubated with the horseradish peroxidase conjugated secondary antibody (goat anti-rabbit IgG; ab205718; Abcam, 1:1000) for 1 h at room temperature. Chemiluminescence detection was performed by chemiluminescence kit (Pierce Chemical, Rockford, USA).
5
Quantifying Protein Expression Levels
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6
Western Blot Analysis of Osteogenic Markers
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Cell lysates were obtained using RIPA lysis buffer (Beyotime, Shanghai, China) containing 10 mM phenylmethylsulphonylfluoride as a protease inhibitor (PMSF; Beyotime) and 50 µg of total protein was separated in a Bis-Tris polyacrylamide gel and transferred onto a nitrocellulose membrane. The membrane was then incubated in 5% bovine serum albumin (BSA) containing primary rabbit-anti-human polyclonal antibodies at 4 °C overnight. After incubating with horseradish peroxidase (HRP)-conjugated goat-anti-rabbit antibody at room for 1 h, protein was detected using electrochemiluminescence (ECL; Millipore, Darmstadt, Germany). The following primary rabbit-anti-human antibodies were used: anti-FOXO1 (1:1000; ab39670, Abcam, Cambridge, MA, USA); anti-GDF5 (1:1000; ab93855, Abcam); anti-SOX6 (1:1000; ab30455, Abcam); anti-Runx2 (1:1000; ab23981, Abcam); anti-Sp7/Osterix (1:2000; ab22552, Abcam); anti-ALP (1:2000; ab95462, Abcam); anti-OCN (1:500; ab93876, Abcam); anti-OPN (1:1000; ab8448, Abcam); and anti-GAPDH (1:2500; ab9485, Abcam). The results of Western blots were quantified using image-J software (http://imagej.net ) analysis.
7
ChIP-qPCR Analysis of SOX6 and OCT4 Binding
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Cellular chromatin was immunoprecipitated with an antibody against SOX6 (ab30455, Abcam), OCT4 (ab19857, Abcam), or IgG (control, Santa Cruz Biotechnology) using the Pierce Agarose ChIP Kit (Pierce). The amount of immunoprecipitated DNA was quantified using Takara SYBR Premix Ex Taq II (Takara). The results are presented as the fold enrichment over the IgG control. The human c-Myc [27 (link)] and miR-125b [32 (link)] promoters were used as positive controls for SOX6 and OCT4 binding, respectively.
8
Identification of SOX6 Interacting Proteins
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Anti-FLAG M2 affinity gel immunoprecipitates from FLAG-SOX6 overexpressing K562 cells were separated on SDS-PAGE and stained with Coomassie brilliant blue (CBB). Protein bands of interest were excised and subjected to LC–MS/MS analysis. Immunoprecipitation, immunoblotting, and Glutathione S-transferase (GST) pulldown assays were performed as described previously (12 (link),33 (link)). For the SOX6/NonO interaction studies, anti-FLAG M2 agarose (Sigma) was incubated with nuclear extracts in lysis buffer containing 50 mM Na-HEPES, 150 mM NaCl, pH 7.5, 10% glycerol, 1 mM EDTA, 0.5 mM DTT, 0.5% Triton X-100, 1 mM PMSF, and a protease inhibitor cocktail (Sigma) from K562 cells to pull down the FLAG-tagged protein and associated proteins. Specifically bound proteins from stringently washed beads with lysis buffer were visualized by western blot with anti-NonO or anti-FLAG antibody after SDS-PAGE. Antibodies used were: NonO (Millipore, 05–950; or validated homemade polyclonal antibody), SOX6 (Abcam, ab30455), γ-globin (Abcam, ab137096), BCL11A (Abcam, ab19487), HA (Roche, 12CA5), FLAG (Proteintech, 20543-1-AP), GAPDH (ABclonal, AC002).
9
Multi-Staining and Immunohistochemistry of Musculoskeletal Tissues
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Serial 5-mm-thick sections were prepared from paraffin-embedded specimens for staining.
Hematoxylin-eosin staining was performed in an autostainer machine (ST5010 XL; Leica Microsystems, Mannheim, Germany) using standard procedures.
Elastic Fibers staining kit for ligament, Alcian Blue staining kit for cartilage and Fast Green staining kit for bone were purchased from Leagene Biotech (DC0066; DB0060; DZ0046).
Sections for immunohistochemical staining were carried out as described previously [49 (link)]. The primary rabbit anti-human antibodies were: anti-FOXO1 (1:200; ab39670, Abcam); anti-GDF5 (1:200; ab93855, Abcam); anti-SOX6 (1:200; ab30455, Abcam).
Hematoxylin-eosin staining was performed in an autostainer machine (ST5010 XL; Leica Microsystems, Mannheim, Germany) using standard procedures.
Elastic Fibers staining kit for ligament, Alcian Blue staining kit for cartilage and Fast Green staining kit for bone were purchased from Leagene Biotech (DC0066; DB0060; DZ0046).
Sections for immunohistochemical staining were carried out as described previously [49 (link)]. The primary rabbit anti-human antibodies were: anti-FOXO1 (1:200; ab39670, Abcam); anti-GDF5 (1:200; ab93855, Abcam); anti-SOX6 (1:200; ab30455, Abcam).
10
Multiparametric Immunofluorescence Analysis of Murine Tissues
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The following primary antibodies were used for FACS and IF staining with murine tissues: Ter-119 (TER-119, Biolegend), CD45 (30-F11, Biolegend), EpCAM (G8.8, Santa Cruz Biotechnology), Pdpn (8.1.1, Biolegend), CD31 (390, Biolegend), Procr (eBio1560, Invitrogen), CD90 (53–2.1, Biolegend), CD55 (RIKO-3, Biolegend), αSMA (1A4, Sigma Aldrich), Adamdec1 (Origine #TA323936), Pcolce2 (Proteintech #10607-I-AP), C3 (11H9, Abcam), Sox6 (Abcam #ab30455), ER-TR7 (Abcam #ab51824), Collagen I (Abcam #ab34710), Collagen VI (Abcam #ab6588), and Fibronectin (Abcam #ab2413). The following secondary antibodies were used: donkey anti-rabbit PE (Poly4064, Biolegend), Alexa Fluor 488-, 546-, 568-, and 647-conjugated secondary antibodies were obtained for goat anti-rabbit, goat anti-rat, goat anti-mouse, and goat anti-Syrian hamster from Life Technologies, and DyLight 488- and 649-conjugated secondary antibodies for goat anti-Syrian hamster were obtained from Biolegend. NucBlue viability dye (Invitrogen) was spiked into single-cell suspensions before flow analysis or FACS sorting.
The following probes from Advanced Cell Diagnostics were used for FISH in murine tissues: Pi16 (Mm-Pi16-C2), Grem1 (Mm-Grem1-C3), and Agt (Mm-Agt-C1).
The following probes from Advanced Cell Diagnostics were used for FISH in murine tissues: Pi16 (Mm-Pi16-C2), Grem1 (Mm-Grem1-C3), and Agt (Mm-Agt-C1).
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