Cells were lysed in modified RIPA buffer (50mM Tris-HCl pH 7.4, 150mM NaCl, 1% IGEPAL, protease and phosphatase inhibitors) and 10–30μg of total protein was loaded into 10% SDS-PAGE gels and transferred to PVDF membranes for Western blotting with the following primary antibodies and dilutions: mouse monoclonal anti-Pax7, 1:10; mouse monoclonal (F5D) anti-myogenin (Developmental Studies Hybridoma Bank, USA), 1:5; mouse monoclonal anti-tubulin, 1:10000; mouse monoclonal anti-HA HRP conjugated, 1:4000 (Sigma-Aldrich,USA); mouse monoclonal anti-HDAC2 [3F3], 1:5000; rabbit polyclonal ChiP-grade anti-HA tag, 1:10000; rabbit polyclonal anti-Nedd4, 1:10000; rabbit monoclonal anti-GFP E385, 1:5000 (Abcam, UK); mouse monoclonal anti-GAPDH (EMD-Millipore, USA), 1:10000; mouse monoclonal 9B11 myc-tag (Cell Signaling, USA), 1:1000; rabbit polyclonal anti-GST (gift from Dr. María Paz Marzolo, Santiago, Chile), 1:5000 and mouse monoclonal P4D1 anti-ubiquitin (Santa Cruz Biotechnology, USA), 1:500. As secondary antibodies HRP conjugated anti-mouse IgG and anti-rabbit IgG (Cell Signaling, USA) were used at 1:5000. HRP activity was detected using the SuperSignal West Dura Extended Duration Substrate (Thermo-Fisher Scientific, USA). When indicated, lysates/fractions were subjected to co-immunoprecipitation as described [16 (link)].
Mouse monoclonal anti pax7
Manufactured by Developmental Studies Hybridoma Bank
Sourced in United States
Mouse monoclonal anti-Pax7 is a laboratory-produced antibody that specifically recognizes the Pax7 protein in mouse samples. Pax7 is a transcription factor involved in the regulation of muscle stem cell development and function.
Automatically generated - may contain errors
Lab products found in correlation
Mouse monoclonal anti gapdh, by Merck Group (2 mentions)
Rabbit monoclonal anti gfp e385, by Abcam (2 mentions)
Supersignal west dura extended duration substrate, by Thermo Fisher Scientific (2 mentions)
Ix71 microscope, by Olympus (2 mentions)
Anti rabbit igg, by Cell Signaling Technology (1 mentions)
Hrp conjugated anti mouse igg, by Cell Signaling Technology (1 mentions)
Mouse monoclonal anti tubulin, by Merck Group (1 mentions)
Alexa fluor 488 goat anti mouse igg, by Jackson ImmunoResearch (1 mentions)
Axio observer z1, by Zeiss (1 mentions)
Anti myogenin m 225, by Santa Cruz Biotechnology (1 mentions)
Anti nedd4, by Abcam (1 mentions)
Alexa 488, by Thermo Fisher Scientific (1 mentions)
Donkey anti rat 488, by Thermo Fisher Scientific (1 mentions)
Goat anti mouse alexa 594, by Thermo Fisher Scientific (1 mentions)
Goat anti mouse alexa 488, by Thermo Fisher Scientific (1 mentions)
Goat anti rabbit alexa 594, by Thermo Fisher Scientific (1 mentions)
Hoechst 33342, by Thermo Fisher Scientific (1 mentions)
Vectashield, by Vector Laboratories (1 mentions)
Evos fl, by Thermo Fisher Scientific (1 mentions)
Rat anti brdu, by Bio-Rad (1 mentions)
6 protocols using mouse monoclonal anti pax7
1
Western Blotting of Muscle Differentiation Markers
2
Quantifying Satellite Cell Differentiation
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Satellite cell cultures and LD muscle sections were immunostained to determine myotube formation and FCA, respectively. Satellite cells were analyzed for purity after isolation and for the expression of the contractile protein myosin heavy chain (MyHC) after 48 h of differentiation. Satellite cells were prefixed and nursery LD muscle sections were postfixed in 4% paraformaldehyde and permeablized with Triton X‐100. Samples were blocked with 10% goat serum in PBST (0.1% Tween‐20 in PBS) for 1 h at room temperature. Cells and slides were incubated overnight at 4°C with the primary antibodies mouse monoclonal anti‐Pax7 at 15 μg/mL (Developmental Studies Hybridoma Bank, Iowa City, IA) and mouse monoclonal anti‐MyHC at 10 μg/mL (Roche) or anti‐dystrophin at 5 μg/mL (R&D Systems, Minneapolis, MN), respectively. Primary antibodies were removed and incubated with the secondary antibody (AlexaFluor 488 goat anti‐mouse IgG at 1:500 dilution, Jackson Immunoresearch) in 5% goat serum for 1 h at room temperature. Myotube formation and FCA images were collected with Zeiss AxioObserver Z.1 and analyzed with ZenPro automated image analysis suite (Carl Zeiss AG).
3
Immunofluorescence Staining of Muscle Markers
4
Immunofluorescent Analysis of Muscle Stem Cells
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Cells were fixed in 4% paraformaldehyde in PBS, permeabilized with 0.5% Triton X-100 and blocked with 3% BSA in PBS. Primary antibodies and dilutions were used as follows: mouse monoclonal anti-Pax7 (Developmental Studies Hybridoma Bank) at 1:5; rat monoclonal anti-MyoD (5F11) (Millipore) at 1:100; mouse monoclonal anti-myogenin (F5D) (Developmental Studies Hybridoma Bank) at 1:5; chicken anti-Syndecan-4 [23 (link)] at 1:500; rabbit monoclonal anti-active caspase-3 (C92-605) (BD Pharmingen) at 1:100. The following secondary antibodies were used at 1:500: goat anti-mouse Alexa 594, goat anti-rabbit Alexa 594, goat anti-mouse Alexa 488, donkey anti-rat 488 (Life technologies) and donkey anti-chicken-AMCA (Jackson). Cell nuclei were stained with Hoechst 33342 (Life technologies) and Vectashield (Vector Laboratories) was used as mounting media. Images were acquired using an IX71 microscope (Olympus) equipped with a QICam FAST QImaging camera and MoticBA410 microscope with a Moticam Pro 252B camera. Images were processed using Illustrator (Adobe).
5
Immunostaining of Myosin Heavy Chain and Pax7
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For detection of myosin heavy chain (MyHC) and Pax7, fixed cells were permeabilized with PBS + 0.2% TritonX100 for 10 min at room temperature. For BrdU immunostaining, after permeabilization the DNA was denatured with 3 N HCl for 20 min at RT followed by a 10 min incubation with 0.1 M sodium borate, pH 8.5 and then three washes in PBS. For all immunostaining procedures, samples were then blocked with 3% bovine serum albumin (BSA) for 1 h before incubation with primary antibodies over night at 4 °C in PBS + 1% BSA. Primary antibodies used were: mouse monoclonal anti-MyHC (Developmental Studies Hybridoma Bank at Iowa University, MF20 clone) at 1:200, mouse monoclonal anti-Pax7 (Developmental Studies Hybridoma Bank at Iowa University) at 1:100 and rat anti-BrdU (Serotec) at 1:100. Cells were then washed 3 times with PBS + 0.2% TritonX100 and incubated for 1 h at room temperature with PBS + 1% BSA + secondary antibodies conjugated with AlexaFluor 488 (Invitrogen) at 1:500 followed by incubation with 2 μg/ml DAPI (Life Technologies) in PBS for 5 min at room temperature. Cells were then washed 3 times with PBS + 0.2% TritonX100 then stored at 4 °C in PBS until imaging on an epifluorescence microscope (EVOS-FL, Life Technologies) using a 10 × objective.
6
Western Blot Analysis of Cell Extracts
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Whole cell extracts were obtained by disruption in modified RIPA lysis buffer (50 mM Tris-HCl pH 7.4, 150 mM NaCl, 1% IGEPAL, protease and phosphatase inhibitors (Merck) and incubated at 4°C for 20 min, followed by centrifugation at 15200 rpm for 10 min. Proteins (20–30 μg) were separated into 10% SDS-PAGE gels and transferred to polyvinylidene difluoride (PVDF) membranes (Thermo Fisher Scientific). Membranes were blocked with 5% (wt/vol) nonfat powdered milk in TBS-T (20 mM Tris, pH 7.4; 100 mM NaCl; 1% Tween-20) and incubated with the following primary antibodies and dilutions: mouse monoclonal anti-Pax7 (Developmental Studies Hybridoma Bank) at 1:10; mouse monoclonal anti-myc-tag (9B11) (Cell Signaling) at 1:1000; rabbit monoclonal anti-GFP (E385) (Abcam) at 1:10000, mouse monoclonal anti-GAPDH (EMD Millipore) at 1:10000; rabbit MultiMab™ anti-phospho-CK2 Substrate (pS/pT)DXE (Cell Signaling) at 1:1000. Anti-mouse IgG and anti-rabbit IgG HRP-conjugated secondary antibodies (Cell Signaling) were used at 1:5000, and HRP activity was visualized using SuperSignal West Pico Chemiluminescent Substrate or SuperSignal West Dura Extended Duration Substrate (Thermo Fisher Scientific). Relative densitometry was measured using ImageJ software.
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