The distal end of nerve and middle part of gastrocnemius muscle was harvested two weeks after various treatment and proteins were extracted. Proteins (50 μg) were resolved by SDS-polyacrylamide gel electrophoresis and transferred onto a blotting membrane. Antibodies against Bcl-2 (1:1000, BD), Bad (1:1000, Santa Cruz), Bax (1:1000, Santa Cruz), 8-oxo-dG (1:500, R&D), desmin (1:200, Proteintech), acetylcholine receptor (1:200, Millipore), GAPDH (1: 1000, Santa Cruz) were incubated overnight at 4°C. AFS and fibroblast cell lysated protein (50 μg) was separated by SDS-PAGE electrophoretically transferred to nitrocellulose membranes. After blocking, the blots were incubated with antibodies for NT-3 (1:1000, Millipore), BDNF (1:1000, Abcam), CNTF (1:1000, Abbiotec), GDNF (1:1000, Abbiotec), and GAPDH (1:1000, Santa Cruz). Horseradish peroxidase-conjugated secondary antibodies were used. Protein bands were analyzed by the ISI1000 image system (Alpha Innotech Corporation, CA, USA).
Desmin
Manufactured by Proteintech
Sourced in United States
Desmin is a structural protein that is primarily found in muscle cells. It plays a crucial role in maintaining the integrity and organization of the cytoskeleton, which is responsible for the shape and mechanical properties of the cell. Desmin is commonly used as a marker for the identification and characterization of muscle tissue and muscle-related disorders.
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Lab products found in correlation
Acetylcholine receptor, by Merck Group (2 mentions)
Brain derived neurotrophic factor (bdnf), by Abcam (2 mentions)
8 oxo dg, by R&D Systems (2 mentions)
Bcl2 associated x (bax), by Santa Cruz Biotechnology (1 mentions)
Gapdh, by Santa Cruz Biotechnology (1 mentions)
Bcl 2, by BD (1 mentions)
Rabbit anti yb 1, by Abcam (1 mentions)
Alexa fluor 488 donkey anti rabbit igg, by Thermo Fisher Scientific (1 mentions)
Podocin, by Abcam (1 mentions)
Alexa fluor 594 donkey anti mouse igg, by Thermo Fisher Scientific (1 mentions)
Goat anti synaptopodin, by Santa Cruz Biotechnology (1 mentions)
Nephrin, by R&D Systems (1 mentions)
α sma, by Novus Biologicals (1 mentions)
Gapdh, by Abcam (1 mentions)
E cadherin, by Merck Group (1 mentions)
Vimentin, by Proteintech (1 mentions)
P lats1, by Affinity Biosciences (1 mentions)
P yap, by Affinity Biosciences (1 mentions)
P taz, by Affinity Biosciences (1 mentions)
α sma, by Abcam (1 mentions)
7 protocols using desmin
1
Protein Expression Analysis of Nerve and Muscle
2
Immunofluorescence Staining of Podocyte Markers
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Antibodies used in this study were as follows: Goat anti-synaptopodin (Santa Cruz), Rabbit anti-YB-1 (Abcam), Rabbit anti-PP2A (Abcam), Alexa Fluor 488 donkey anti-rabbit IgG (Life Technologies), Alexa Fluor 594 donkey anti-mouse IgG (Life Technologies), BMP7 (Abcam), a-SAM (Abcam), Desmin (Proteintech), ZO-1 (Proteintech), WT-1 (Abcam), Nephrin (R&D Systems), Podocin (Abcam).
3
Immunocytochemical Characterization of HSCs
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HSCs were grown on chamber slides, and the transfection experiments were carried out as described before. The cells were fixed in 4% paraformaldehyde for 15 min and washed with PBS three times. The cells were permeated with X-Triton100 for 20 min and washed with PBS three times. Then the cells were blocked with 5% bovine serum albumin in PBS for 1 h followed by incubation with primary antibodies against desmin (ProteinTech Group, Chicago, USA) and α-SMA (Novus Biologicals, Littleton, USA) for 16 h at 4 °C overnight. After washing with PBS three times, secondary antibody was applied and incubated for 1 h. After additional washing, the cells were analyzed by fluorescence microscopy.
4
CCl4 and TGF-β1 Induced Liver Fibrosis
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The CCl4 and TGF-β1 were purchased from Sigma (St Louis, MO, USA). Antibodies against type I collagen (Affinity, NO. AF7001, 1:1000), α-SMA (Abcam, NO. ab32575, 1:2000), E-cadherin (Sigma, NO. MABT26, 1:1000), BMP-7 (Sigma, NO. MAB4350, 1:1000), Desmin (Proteintech, NO. 16520-1-AP, 1:50000), Vimentin (Proteintech, NO. 60330-1-Ig, 1: 50000), YAP (Proteintech, NO. 13584-1-AP, 1: 5000), TAZ (Proteintech, NO. 23306-1-AP, 1:2000), p-YAP (Affinity, NO. AF3328, 1:1000), p-TAZ (Affinity, NO. AF4315, 1:1000), MST1 (Affinity, NO. DF8430, 1:2000), p-MST1 (Affinity, NO. AF3688, 1:1000), LATS1 (Affinity, NO. AF7669, 1:1000), p-LATS1 (Affinity, NO. AF7169, 1:1000), NF2 (Abcam, NO. ab109244, 1:100000) and GAPDH (Abcam, NO. ab9485, 1:2500) were purchased. Adenoviral vectors expressing the scrambled shRNA (Ad-shCtrl), Ad-shSNHG5, adenoviral vectors expressing a control scrambled sequence (Ad-Ctrl), Ad-SNHG5 and adenoviral vectors expressing NF2 (Ad-NF2) were purchased from Genechem Co.Ltd (Shanghai, China). Animal experimental protocols were approved by the University Animal Care and Use Committee after mice were supplied by the Experimental Animal Center of Wenzhou Medical University.
5
Immunohistochemical Analysis of Nerve and Muscle Regeneration
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Sciatic nerve and gastrocnemius muscle were subjected to immunohistochemistry with antibodies against NT-3 (1:200, Millipore), BDNF (1:200, Abcam), CNTF (1:200, Abbiotec), GDNF (1:200, Abbiotec), S-100 (1:200, Serotec), neurofilament (1:200, Millipore), desmin (1:200, Proteintech), acetylcholine receptor (1:200, Millipore) and 8-oxo-dG (1:500, R&D) for detection of nerve and muscle regeneration. AF 488 donkey anti-mouse IgG and AF594 donkey anti-rabbit (Invitrogen; 1:200 dilutions) were used to stain and viewed by confocal microscopy capture image. Five consecutive resections (approximately maximum diameter) were chosen and measured by the IS 1000 image analysis system according to our previous study [31 (link)]. The number of cells was counted in randomly selected 20 squares from100 squares in an ocular grid. The region of maximum diameter of the resected nerve tissue and muscle was chosen to be examined. Areas of activities (0.2 mm2) appeared dense against background and were measured.
6
Extracellular Matrix and Cell Characterization
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The dECM underwent fixation in 4% paraformaldehyde (Richjoint, Shanghai, China), followed by PBS washing, permeabilization with 1% Triton X-100 (Richjoint, Shanghai, China) for 15 min, and blocking in 5% FBS (Hyclone) for additional 15 minutes. Subsequently, the fixed dECM was subjected to incubation with appropriately diluted primary antibodies against type I collagen (Proteintech), type III collagen (Proteintech), fibronectin (Proteintech), and laminin (Abcam). Following a PBS rinse, the dECM was then incubated with Cy3-labelled goat anti-mouse IgG (Proteintech). Fluorescence images were captured using a confocal microscope (Leica, TCS SP5).
For intracellular staining, ADSCs underwent PBS washing, followed by permeabilization with 1% Triton X-100 (Richjoint, Shanghai, China) for 30 min and subsequent blocking in 5% FBS (Hyclone) for 5 min. Subsequently, the cells were subjected to staining with primary antibodies against MHC (Thermo Fisher Scientific), myosin (Proteintech), and desmin (Proteintech), followed by Cy3-labelled goat anti-mouse IgG (H + L). Finally, Hoechst stain (Beyotime Biotechnology) was applied to the cells, and the observation was conducted using a confocal microscope (Leica, TCS SP5).
For intracellular staining, ADSCs underwent PBS washing, followed by permeabilization with 1% Triton X-100 (Richjoint, Shanghai, China) for 30 min and subsequent blocking in 5% FBS (Hyclone) for 5 min. Subsequently, the cells were subjected to staining with primary antibodies against MHC (Thermo Fisher Scientific), myosin (Proteintech), and desmin (Proteintech), followed by Cy3-labelled goat anti-mouse IgG (H + L). Finally, Hoechst stain (Beyotime Biotechnology) was applied to the cells, and the observation was conducted using a confocal microscope (Leica, TCS SP5).
7
Immunofluorescence Staining of Muscle Markers
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With immunofluorescence staining, the expression of markers was detected using antibodies against desmin (Proteintech), PAX7 (Proteintech), MYHC (Thermo Fisher Scientific), MYOD (Invitrogen), F-actin (Abcam) and dystrophin (Santa Cruz Biotechnology, TX, USA). Briefly, cell samples were washed thrice with PBS and then fixed with 4% paraformaldehyde for 15 min. After washing with PBS, the cells were permeabilized with 0.25% Triton X-100 (Rich Joint, Shanghai, China) for 10 min and incubated in 5% fetal bovine serum at 37°C for 1 h. Afterward, cells were incubated with primary antibodies overnight. After washing, secondary antibody was used to incubate the cells for 1 h at room temperature. The cells were then washed and treated with Hoechst (Beyotime) to stain the cell nuclei. In the end, samples were imaged under a fluorescence microscope (Olympus). Using ImageJ software according to the method described by Menconi et al. [Citation32, Citation33] , quantitative image analysis was performed by analyzing five randomly selected injury fields per sample; fusion index was calculated as the number of nuclei in myotubes divided by the total number of nuclei counted. The average number of nuclei per myotube was determined by dividing the number of nuclei in myotubes by the total number of myotubes.
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