Cells were lysed with RIPA buffer (Sigma-Aldrich) containing protease inhibitor cocktail (Fisher Scientific, Waltham, MA, USA) or fixed with 4% paraformaldehyde (PFA), then permeabilized with 0.1% Triton X-100. Proteins transferred to nitrocellulose membranes were probed with the indicated antibodies against: ASM (Abcam, Cambridge, MA, USA), desmin (Santa Cruz, Dallas, TX, USA), dysferlin (Novocastra, Buffalo Grove, IL, USA), myogenin (Dako, Carpinteria, CA, USA), GAPDH (Santa Cruz), myosin heavy chain 3 (Developmental Studies Hybridoma Bank, Iowa City, IA, USA), and α-actinin (Epitomics, Burlingame, CA, USA). Primary antibodies were followed by the appropriate HRP-conjugated secondary antibodies (Sigma-Aldrich), and chemiluminescent western blotting substrate (Fisher, Waltham, MA, USA; GE Healthcare, Pittsburgh, PA, USA) then processed on Bio-Lite X-ray film (Denville Scientific, Metuchen, NJ, USA). For immunostaining, permeabilized cells were reacted with anti-dysferlin (Epitomics) and anti-LAMP1 (Santa Cruz) antibodies, followed by fluorophore-conjugated secondary antibodies: Alexa Fluor 488-anti-rabbit, Alexa Fluor 594-anti-rat, and Alexa Fluor 594-anti-mouse (Life Technologies). Nuclei were counterstained with Hoechst 33342. After mounting in mounting medium (Dako), cells were imaged as described in Supplementary Methods.
Dysferlin
Manufactured by Leica
Sourced in United States, United Kingdom
Dysferlin is a specialized laboratory equipment designed for the analysis and detection of the dysferlin protein. It is a crucial tool in the study of muscular dystrophy and other related neuromuscular disorders. The primary function of Dysferlin is to provide accurate and reliable measurements of the dysferlin protein levels in biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Hrp conjugated secondary antibody, by Merck Group (2 mentions)
Ripa buffer, by Merck Group (2 mentions)
Chemiluminescent western blotting substrate, by Thermo Fisher Scientific (2 mentions)
Protease inhibitor cocktail, by Thermo Fisher Scientific (2 mentions)
Bio lite x ray film, by Thomas Scientific (2 mentions)
Calpain 3, by Leica (2 mentions)
β sarcoglycan, by Leica (2 mentions)
α sarcoglycan, by Leica (2 mentions)
γ sarcoglycan, by Leica (2 mentions)
δ sarcoglycan, by Leica (2 mentions)
α dystroglycan, by Merck Group (2 mentions)
Alexa fluor 594 anti rat, by Thermo Fisher Scientific (1 mentions)
Mounting medium, by Agilent Technologies (1 mentions)
Myogenin, by Agilent Technologies (1 mentions)
Desmin, by Santa Cruz Biotechnology (1 mentions)
Alexa fluor 488 anti rabbit, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 594 anti mouse, by Thermo Fisher Scientific (1 mentions)
Anti lamp1, by Santa Cruz Biotechnology (1 mentions)
Gapdh, by Santa Cruz Biotechnology (1 mentions)
Caveolin 3, by BD (1 mentions)
7 protocols using dysferlin
1
Western Blot and Immunostaining of Skeletal Muscle Proteins
2
Comprehensive Muscle Protein Analysis Protocol
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The proband underwent a muscle biopsy after giving written informed consent, according to Institutional guidelines. Morphological examination was performed according to standard procedures.
Immunohistochemical (IHC) analyses were performed using monoclonal antibodies directed against three different epitopes of dystrophin (rod-domain diluted 1:10, NH2-domain diluted 1:10, COOH-domain undiluted, all from Novocastra, Newcastle upon Tyne, UK), sarcoglycans (alpha-sarcoglycan 1:20, gamma-sarcoglycan 1:10, Novocastra, Newcastle upon Tyne, UK), and caveolin-3 (1:1000, BD Transduction Laboratories, Franklin Lakes, NJ, USA) as previously described [23 (link)].
Forty micrograms of muscle protein lysates was probed for calpain-3 (Novocastra, 2C4, 1:100), dysferlin (Novocastra, Newcastle upon Tyne, UK, Hamplet-2 1:1000), dystrophin (rod-domain 1:200 and COOH-term 1:80), actinin (Sigma, St. Louis, MO, USA, 1:10,000), and sarcoglycans (alpha-sarcoglycan 1:280, beta-sarcoglycan 1:65, gamma-sarcoglycan 1:200, delta-sarcoglycan 1:60, all from Novocastra) expressions by Western blot (WB) analysis on a 10% polyacrylamide gel.
Immunohistochemical (IHC) analyses were performed using monoclonal antibodies directed against three different epitopes of dystrophin (rod-domain diluted 1:10, NH2-domain diluted 1:10, COOH-domain undiluted, all from Novocastra, Newcastle upon Tyne, UK), sarcoglycans (alpha-sarcoglycan 1:20, gamma-sarcoglycan 1:10, Novocastra, Newcastle upon Tyne, UK), and caveolin-3 (1:1000, BD Transduction Laboratories, Franklin Lakes, NJ, USA) as previously described [23 (link)].
Forty micrograms of muscle protein lysates was probed for calpain-3 (Novocastra, 2C4, 1:100), dysferlin (Novocastra, Newcastle upon Tyne, UK, Hamplet-2 1:1000), dystrophin (rod-domain 1:200 and COOH-term 1:80), actinin (Sigma, St. Louis, MO, USA, 1:10,000), and sarcoglycans (alpha-sarcoglycan 1:280, beta-sarcoglycan 1:65, gamma-sarcoglycan 1:200, delta-sarcoglycan 1:60, all from Novocastra) expressions by Western blot (WB) analysis on a 10% polyacrylamide gel.
3
Dysferlin and Annexin A2 Western Blot
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Cells were lysed with RIPA buffer (Sigma-Aldrich) containing a protease inhibitor cocktail (Fisher Scientific, Waltham, MA, USA). Proteins transferred to nitrocellulose membranes were probed with the indicated antibodies against dysferlin (Novocastra, Buffalo Grove, IL, USA), Anx A2 (BD biosciences, #610068), caveolin-1 (Abcam, #ab2910), b-actin (Cell Signaling, #4967), or cadherin (Cell Signaling, #4068). Primary antibodies were followed by the appropriate HRP-conjugated secondary antibodies (Sigma-Aldrich) and chemiluminescent western blotting substrate (Fisher, Waltham, MA, USA; GE Healthcare, Pittsburgh, PA, USA). The blots were then processed on Bio-Lite X-ray film (Denville Scientific, Metuchen, NJ, USA), and signals for AnxA2 and dysferlin protein bands were normalized to that of the internal control‒‒cadherin.
4
Muscle Biopsies and Immunohistochemical Analysis
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Muscle biopsies were performed in three patients, and one patient had been examined previously at other hospital. Three muscle specimens obtained at our center were taken from the biceps brachii (ID185), paraspinal muscle (ID37), and vastus lateralis (ID131). Frozen muscle sections (5-µm thick) obtained from all muscle specimens were stained with hematoxylin and eosin (H&E), modified Gomori trichrome (modified GT), and reduced nicotinamide adenine dinucleotide-tetrazolium reductase (NADH-tr). Muscle specimens were also analyzed by immunohistochemistry using antibodies against the C-terminus, rod, and N-terminus of dystrophin (Leica Microsystems, Newcastle upon Tyne, UK), dysferlin (Leica Microsystems), α-sarcoglycan (Leica Microsystems), β-sarcoglycan (Leica Microsystems), γ-sarcoglycan (Leica Microsystems), δ-sarcoglycan (Leica Microsystems), α-dystroglycan (Millipore, Billerica, MA, USA), and caveolin (BD Biosciences, San Diego, CA, USA).
5
Muscle Protein Analysis in Patients
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Muscle specimens were obtained for nine patients, taken from the vastus lateralis (F1, F4, F5c, F7, F9, and F10) and the biceps brachii (F5b, F6, and F8). Frozen muscle sections (5-µm thickness) from all muscle specimens were stained with hematoxylin and eosin, modified Gomori trichrome, and reduced nicotinamide adenine dinucleotide-tetrazolium reductase. Protein analysis was performed in seven patients (F4, F5b, F6, F7, F8, F9, and F10). Western blots used antibodies against calpain-3 (Novocastra, NewcastleuponTyne, UK). Muscle specimens were also analyzed by immunohistochemistry using antibodies against the C-terminus of dystrophin (Leica Microsystems, Newcastle upon Tyne, UK), the rod domain of dystrophin (Leica Microsystems), the N-terminus of dystrophin (Leica Micro-systems), dysferlin (Leica Microsystems), α-sarcoglycan (Leica Microsystems), β-sarcoglycan (Leica Microsystems), γ-sarco-glycan (Leica Microsystems), δ-sarcoglycan (Leica Microsystems), α-dystroglycan (Millipore, Billerica, MA, USA), and caveolin (BD Biosciences, San Diego, CA, USA).
6
Comprehensive Muscle Biopsy Analysis
7
Muscle Biopsy Immunostaining Protocol
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Muscle biopsy cryosections of 6-μm thickness were immunostained using commercially available antibodies against dystrophin, dysferlin, alpha-, beta-, gamma, and delta-sarcoglycans (Leica, IL, USA), alpha-dystroglycan (Merk Millipore, MA, USA) according to standard protocols with a Ventana Benchmark automated stainer [15 (link)].
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