MDSPCs and BM-MSCs at passages 4-5 were tested for CD34, CD45, CD90, c-kit, and desmin by immunofluorescence staining. All antibodies used for immunofluorescence staining were purchased from Abcam, and desmin was purchased from Sigma-Aldrich. Before staining, the cells were rinsed in PBS, fixed in 2% formaldehyde (Sigma-Aldrich; 10% formalin diluted in PBS) for 15 min, and rinsed again. Before antibody incubation, cells were blocked with either 10% horse serum or 10% goat serum for 1 hour, to permeabilise the cells and block nonspecific protein-protein interactions. Afterwards, the cells were incubated with primary antibody in PBS overnight at 4°C, followed by incubation with secondary antibody in PBS for 1 hour at room temperature, followed by incubation with streptavidin-Cy3 1 : 400 in PBS for 15 min (only for CD34, c-kit, and desmin), and counterstained with 4,6-diamidino-2-phenylindole dihydrochloride hydrate (DAPI; Sigma-Aldrich) 1 : 10,000 in PBS for 10 min. After each step, the cells were rinsed in PBS.
Desmin
Manufactured by Merck Group
Sourced in United States, United Kingdom
Desmin is a type of intermediate filament protein found in muscle cells. It plays a crucial role in maintaining the structural integrity and organization of the cytoskeleton within muscle cells.
Automatically generated - may contain errors
Lab products found in correlation
α sma, by Merck Group (5 mentions)
Gapdh, by Merck Group (3 mentions)
Ck 19, by Merck Group (2 mentions)
Glial fibrillary acidic protein (gfap), by Merck Group (2 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (2 mentions)
Alexa fluor 568, by Thermo Fisher Scientific (2 mentions)
Alexa 488, by Thermo Fisher Scientific (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (2 mentions)
Nanog, by Santa Cruz Biotechnology (2 mentions)
Bovine serum albumin (bsa), by Merck Group (2 mentions)
Fast myhc, by Merck Group (1 mentions)
Phospho rictor thr1135, by Cell Signaling Technology (1 mentions)
P70 s6 kinase, by Cell Signaling Technology (1 mentions)
α tubulin, by Merck Group (1 mentions)
Phospho p70 s6 kinase, by Cell Signaling Technology (1 mentions)
Foxo3a, by Cell Signaling Technology (1 mentions)
Phospho mtor ser2448, by Cell Signaling Technology (1 mentions)
Rictor, by Cell Signaling Technology (1 mentions)
Trans blot turbo transfer system, by Bio-Rad (1 mentions)
Enhanced chemiluminescence system, by GE Healthcare (1 mentions)
25 protocols using desmin
1
Immunofluorescence Characterization of MSCs
2
Western Blotting Protocol for Protein Analysis
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Equal protein loading was prepared before resuspending with 2 × Laemmli sample buffer. The mixture was collected by short spin centrifugation and boiled for 5 min before loaded into SDS–polyacrylamide gel electrophoresis. Lysate was separated on SDS–polyacrylamide gel electrophoresis (6, 8, or 15% acrylamide) with 200 V for 1 h. The separated protein was transferred onto the nitrocellulose membrane using the Transblot Turbo® Transfer System (BioRad, UK). The membranes were blocked in 5% reduced-fat milk for 1 h before incubate with primary antibodies. The antibodies are Fast-MyHC, α-tubulin, SSRP-1 (1:1000) and Desmin, (1:2000), from Sigma-Aldrich, UK. PTEN (1:1000) (a gift from Dr. Zubair Ahmed, Medical School, University of Birmingham). PI3K, phospho-PI3K (Tyr458), Akt, phospho-Akt (Ser473), mTOR, phospho-mTOR (Ser2448), p70S6 Kinase, phospho-p70S6 Kinase, Rictor, phospho-Rictor (Thr1135) and FoxO3a (1:1000) from Cell Signalling Technology, UK. Beclin1, Atg5, Atg7, and LC3B (1:1000) (a gift from Dr. Melissa Grant, School of Dentistry, University of Birmingham).
3
Comprehensive Antibody Staining Protocol
4
Western Blot Analysis of Signaling Proteins
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The total protein was extracted from the cells with lysis buffer containing protease inhibitors (Roche, UK). The protein concentration was measured by a BCA-200 protein assay kit (Pierce, USA). Equal amounts of proteins were separated by 4-12% sodium dodecyl sulfate/polyacrylamide gel electrophoresis and transferred to a polyvinylidene fluoride (PVDF) membrane. The membrane was blocked in TRIS-buffered saline with Tween (TBST) containing 5% fat-free milk for 2 h and probed with primary antibodies, p-GSK3β (1 : 1000; Cell Signaling), t-GSK3β (1 : 1000; Cell Signaling), active β-catenin (1 : 1000; Cell Signaling), myosin (1 : 500; Sigma), α-SMA (1 : 500; Sigma), desmin (1 : 500, Sigma), and GAPDH (1 : 1000; Cell Signaling) overnight at 4°C and then incubated for 2 h with a horseradish-peroxidase-conjugated anti-mouse IgG antibody or anti-rabbit IgG diluted 1 : 2000 (Cell signaling). Protein bands were visualized on an X-ray film by using an enhanced chemiluminescence system (GE Healthcare, Buckinghamshire, UK). The relative protein expression intensities were quantified by densitometry using Quantity One analysis software.
5
Quantifying Skeletal Muscle Fiber Types
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A second set of hindlimb cross-sections were stained to identify fiber type ratios and fiber size as previously described (Yates et al., 2016 (link)). Briefly, fibers in medial and lateral muscle groups expressing myosin heavy chain (MyHC) isoforms specific for Type I and Type II fibers were identified with antibodies raised in the mouse against MyHC-I (BA-D5, 1:20; Developmental Studies Hybridoma Bank (DSHB), University of Iowa, Iowa City, IA) and MyHC-II (F18, 1:25; DSHB), respectively. Muscle sections were counterstained with antibodies raised in the rabbit against desmin (1:1,000, Sigma–Aldrich) to identify all fibers. Immunocomplexes were detected with affinity-purified immunoglobulin antiserum conjugated to Alexa Fluor 594 (1:2,000) or Alexa Fluor 488 (1:1,000). All fluorescent images were visualized on an Olympus IX73 and digital micrographs were captured with a DP80 microscope camera (Olympus, Center Valley, PA). Images were analyzed with Olympus cellsSens Dimension software to determine proportions of positive nuclei or fibers within fetal skeletal muscle sections. Animal identifications and treatments were deidentified prior to analyses.
6
Isolation and Characterization of Primary Human Hepatic Stellate Cells
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A liver cell suspension was prepared using a 2-step collagenase perfusion technique with modifications as described previously.33 (link),34 In brief, hepatocytes were removed by multiple cycles of low-speed centrifugation, which resulted in a supernatant that contained the nonparenchymal cells. phHSCs were subsequently purified from the nonparenchymal cell fraction by discontinuous density centrifugation using Percoll.33 (link) Quality of isolation was checked using immunocytomicrocopy against α-SMA (Sigma-Aldrich), Desmin (Sigma-Aldrich), GFAP (Sigma-Aldrich), and CK-19 (Merck Millipore) (Figure 14 ).![]()
7
Immunofluorescence Analysis of Formalin-Fixed Tissues
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Formalin-fixed, paraffin-embedded tissues were sectioned (5μm thickness), transferred to Superfrost/Plus Microscope Slides (Fisher Scientific), and incubated for 30’ at 60°C. Slides were washed with PBST and blocked with PBS + 5% fetal bovine serum (Sigma-Aldrich) and 3% goat serum (Sigma-Aldrich). Slides were then incubated for 1 hour at room temperature with antibodies specific for albumin (Santa Cruz Biotechnologies), vimentin (Millipore), desmin (Sigma-Aldrich), α-SMA (Sigma-Aldrich), or GFP (Invitrogen). They were then washed in PBST and incubated with combinations of the following secondary antibodies: Cy3 labeled goat anti-rabbit IgG (GE Healthcare), Cy3 labeled anti mouse and Cy3 anti-chicken (Millipore), or Alexa Fluor 488 anti-rabbit (Cell Signaling Technology). After washing in PBST, slides were counterstained with Hoechst 33258 (Molecular Probes) and mounted using Prolong Gold Antifade Reagent (Invitrogen).
8
Multicolor Immunostaining of Zebrafish Embryos
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Embryos were collected, treated with 1-phenyl-2-thiourea (PTU) at 24 hpf to prevent pigmentation, and fixed in 4% PFA for 2 hours at room temperature, after stopping the heart with 0.4% Tricaine to prevent it from collapsing during fixation. After exchanging the fixative with PBS/0.1% Tween washes, yolks were removed using forceps, incubated in 0.1 M glycine for 10 min, and then washed with PBS/1% BSA/1% DMSO/0.5% Triton-X (PBDT), and blocked with PBDT/10% goat serum before incubating in primary antibody at 4°C overnight. The embryos were washed in PBDT and incubated in secondary antibody for 2 hours at room temperature, then incubated with DAPI (2 µg/mL) for 10 min and washed with PBS/0.1% Tween.
Primary antibodies used were GFP (Abcam, 1:800 dilution); N-cadherin (Abcam, 1:250 dilution); p-myosin (Abcam, 1:200); tRFP (Evrogen, 1:200 dilution); Desmin (Sigma, 1:100); and Alcama (DSHB ZN-8, 1:50). α-catenin epitope α-18 (1:300) antibody was a generous gift from Prof. Akira Nagafuchi. Secondary antibodies (1:500 dilution) used were Alexa Fluor 568, Alexa Fluor 488, and Alexa Fluor 647 (Thermo Fisher Scientific).
Primary antibodies used were GFP (Abcam, 1:800 dilution); N-cadherin (Abcam, 1:250 dilution); p-myosin (Abcam, 1:200); tRFP (Evrogen, 1:200 dilution); Desmin (Sigma, 1:100); and Alcama (DSHB ZN-8, 1:50). α-catenin epitope α-18 (1:300) antibody was a generous gift from Prof. Akira Nagafuchi. Secondary antibodies (1:500 dilution) used were Alexa Fluor 568, Alexa Fluor 488, and Alexa Fluor 647 (Thermo Fisher Scientific).
9
Immunohistochemical Analysis of Tissue Samples
10
Immunophenotyping of Muscle Stem Cells
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Cells were fixed in 4% formaldehyde for 10 minutes. Staining for Pax7 (DSHB), desmin (Sigma), and ED1/ ED2 (AbD Serotec) was performed at 4°C for 45 minutes. Appropriate secondary antibodies were applied for 30 minutes at room temperature. Isotype controls were prepared in parallel. Samples were run on a LSRII cytometer and analysed with Facs Diva software (BD Biosciences).
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