To further evaluate the in vitro differentiation capacities of the STRO-1+ hDPSCs and c-Kit+ hAFSCs after their induction toward a myogenic lineage using the protocols outlined above, Western blot (WB) analysis was performed. Whole cell lysates were obtained at 2 weeks after initiating differentiation and processed as previously described by Pisciotta et al. [21 (link)]. Thirty micrograms of protein extract, quantified by a Bradford Protein Assay (Bio-Rad Laboratories, Hercules, CA, USA), underwent SDS-polyacrylamide gel electrophoresis and were transferred to PVDF membranes. The following Abs were used: mouse anti-myogenin and rabbit anti-desmin (Sigma-Aldrich), all diluted 1:1000. Peroxidase-labelled anti-mouse (diluted 1:2000) and anti-rabbit (diluted 1:3000) secondary Abs were used. Whole cell lysate obtained from C2C12 myoblasts was used as a positive control. The membranes were visualized by using enhanced chemioluminescence (Amersham, now part of GE Healthcare, Little Chalfont, UK). Anti-actin Ab was used as control of protein loading. Densitometry of the bands was performed by using ImageJ analysis software (National Institutes of Health, Bethesda, MD, USA). Data were then normalized to values of background and the control actin band.
Rabbit anti desmin
Manufactured by Merck Group
Sourced in Sao Tome and Principe
Rabbit anti-desmin is a primary antibody used in immunohistochemistry and Western blotting applications to detect the presence of desmin, an intermediate filament protein found in muscle cells. This antibody is specific to the desmin protein and can be used to identify and characterize muscle tissue samples.
Automatically generated - may contain errors
Lab products found in correlation
Mouse anti myogenin, by Merck Group (2 mentions)
Bradford protein assay, by Bio-Rad (1 mentions)
Rabbit anti s100b, by Merck Group (1 mentions)
Mouse anti vimentin, by Merck Group (1 mentions)
Mouse anti fibronectin, by Merck Group (1 mentions)
Mouse anti gfp, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (1 mentions)
Rabbit anti 5 ht, by Merck Group (1 mentions)
Rabbit anti runx2, by Abcam (1 mentions)
Dmem low glucose, by Euroclone (1 mentions)
5 protocols using rabbit anti desmin
1
Western Blot Analysis of Myogenic Differentiation
2
Characterization of Immune-Selected hDPSCs
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In order to confirm the phenotype of the immune-selected hDPSCs the expression of the stemness markers STRO-1 and c-Kit, the neural crest-related antigens nestin and SOX10, and the immunomodulatory molecule FasL was investigated. Subsequently, the multipotency of the STRO-1+/c-Kit+ hDPSCs population was demonstrated by culturing cells in appropriate differentiation media to reach osteogenic, myogenic and glial differentiation, respectively, as formerly described (Di Tinco et al., 2021a; Zordani et al., 2019 (link); Carnevale et al., 2018 (link)). At the end of each differentiation experiment, the commitment was evaluated by assessing the expression of lineage related markers with the use of the following primary antibodies: mouse anti-osteocalcin (OCN), rabbit anti-RUNX2 (Abcam), mouse anti-myogenin, rabbit anti-desmin and rabbit anti-S100b (all from Sigma Aldrich). Confocal immunofluorescence analyses were performed as detailed below.
3
Immunophenotyping of GS-1 Cell Line
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Immunophenotyping of the GS-1 cell line was carried out at the 30th passage following the method described previously [10 (link)] with some modifications.
Briefly, the cells were grown on coverslips in six-well plates for 24 hr and then fixed with 4% paraformaldehyde at 4°C for 1 hr and permeabilized with 0.2% Triton-X 100 at room temperature
for 15 min. After blocking with 2% bovine serum albumin (BSA) in PBS at room temperature for 2 hr, the cells were then incubated with four different primary antibodies of
mouse-anti-cytokeratin, mouse-anti-vimentin, mouse-anti-fibronectin and rabbit-anti-desmin (Sigma-Aldrich Co., St. Louis, MO, U.S.A.) at a dilution of 1:200 at room temperature for 2 hr to
characterize the cell line. Subsequently, the cells were rinsed three times with PBS and were incubated with the appropriate secondary antibodies of goat-anti-mouse FITC-conjugated or
goat-anti-rabbit FITC-conjugated (Sigma-Aldrich) at a dilution of 1:320 in PBS containing 1% BSA at room temperature for 1 hr. In control coverslips, only PBS with 1% BSA was used in place
of the primary antibodies. Diaminophenylindole at a final concentration of 1 µg/ml was used to stain DNA in nuclei. All samples were observed with a Carl
Zeiss fluorescence microscope.
Briefly, the cells were grown on coverslips in six-well plates for 24 hr and then fixed with 4% paraformaldehyde at 4°C for 1 hr and permeabilized with 0.2% Triton-X 100 at room temperature
for 15 min. After blocking with 2% bovine serum albumin (BSA) in PBS at room temperature for 2 hr, the cells were then incubated with four different primary antibodies of
mouse-anti-cytokeratin, mouse-anti-vimentin, mouse-anti-fibronectin and rabbit-anti-desmin (Sigma-Aldrich Co., St. Louis, MO, U.S.A.) at a dilution of 1:200 at room temperature for 2 hr to
characterize the cell line. Subsequently, the cells were rinsed three times with PBS and were incubated with the appropriate secondary antibodies of goat-anti-mouse FITC-conjugated or
goat-anti-rabbit FITC-conjugated (Sigma-Aldrich) at a dilution of 1:320 in PBS containing 1% BSA at room temperature for 1 hr. In control coverslips, only PBS with 1% BSA was used in place
of the primary antibodies. Diaminophenylindole at a final concentration of 1 µg/ml was used to stain DNA in nuclei. All samples were observed with a Carl
Zeiss fluorescence microscope.
4
Immunohistochemical Analysis of Zebrafish Larvae
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Zebrafish larvae were anesthetized and fixed in 4% paraformaldehyde (PFA) for 2 h at room temperature (RT). The larvae were washed 3 × 30 min in distilled water27 (link) and then were incubated for 1 h in blocking solution (2% normal goat serum, 1% bovine serum albumin, 1%dimethylsulfoxide, 0.1% Triton X-100 in PBS). The larvae were incubated overnight at RT in primary antibodies diluted in blocking solution. The primary antibodies used were mouse anti-GFP (1: 5000 dilution; Invitrogen), rabbit anti-5-HT (1: 4000 dilution; Sigma-Aldrich) and rabbit anti-Desmin (1:20 dilution; Sigma-Aldrich). The larvae were rinsed extensively in PBS with Triton X-100 (PBST) and incubated overnight at RT in secondary antibodies diluted in blocking solution. The secondary antibodies were Alexa Fluor 488 (1: 500 dilution; Life Technologies) or 564 (1: 500 dilution; Invitrogen). After rinsing in PBST, larvae were transferred to 50% glycerol in PBS.
5
Differentiation of Human Dental Pulp Stem Cells
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Human DPSCs obtained after disaggregation of floating 3D spheres were seeded at cell densities of 3 × 103 cells/cm2 and 4 × 103 cells/cm2, respectively, for the induction toward osteogenic and myogenic lineages. Osteogenic differentiation of hDPSCs was induced for 3 weeks as previously described (Bianchi et al., 2017 (link); Paino et al., 2017 (link)). Cells were cultured in osteogenic medium consisting in α-MEM supplemented with 5% FBS, 100 μM 2P-ascorbic acid, 100 nM dexamethasone, and 10 mM β-glycerophosphate, for 2 weeks. For myogenic induction, hDPSCs were preliminarily treated with the demethylating agent 5-aza-2′-deoxycytidine (10 μM) for 24 h, then differentiation was carried out in DMEM Low Glucose (Euroclone, Italy) plus 5% horse serum, 0.5% chicken serum, 2 mM L -glutamine, 100 U/ml penicillin, 100 μg/ml streptomycin, and 10 nM insulin for 3 weeks, as formerly described (Pisciotta et al., 2015b (link)). At the end of the induction period, confocal immunofluorescence analysis was performed to evaluate the achievement of both the commitments, through the expression of specific tissue related markers. The following primary antibodies were used: mouse anti-osteopontin (OPN), rabbit anti-Runx-2 (Abcam) and rabbit anti-desmin (Sigma-Aldrich).
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