Heart tubes were stained and imaged whole mount to assess the structure. Tubes were fixed in 4% paraformaldehyde (Electron Microscope Sciences) in 1X PBS supplemented with 1:200 Triton-X 100 (9002-93-1, Sigma) for 1 h. Next, tubes were washed 3 times for 30 min each in 1X PBS on a rotary shaker. Samples were then blocked in 5% goat serum in 1× PBS overnight on a shaker followed by washing 3 times for 30 min in 1X PBS. Mouse anti-sarcomeric α-actinin antibody (A7811, Sigma-Aldrich) was diluted to 1:100 in 1× PBS and incubated with the tissues overnight on a shaker. Tubes were then washed 3 times for 30 min each in 1× PBS before incubating with 1:200 DAPI (D1306, Molecular Probes), 3:200 phalloidin conjugated with Alexa-Fluor 488 (A12379, Life Technologies), and 1:100 goat anti-mouse antibody conjugated with Alexa-Fluor 555 (A21422, Life Technologies) in PBS overnight on a shaker. After the final incubation step, samples were rinsed 3 times for 30 min each in 1× PBS. The fluorescently stained heart tubes were imaged with two different confocal microscopes (A1R, Nikon and LSM 700, Zeiss).
Mouse anti sarcomeric α actinin antibody
Manufactured by Merck Group
The Mouse anti-sarcomeric α-actinin antibody is a laboratory reagent used for the detection and localization of sarcomeric α-actinin, a crucial structural protein found in the Z-discs of striated muscle cells. This antibody can be utilized in various immunological techniques, such as immunohistochemistry and Western blotting, to study the distribution and expression of sarcomeric α-actinin in biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Lsm 700, by Zeiss (2 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (2 mentions)
D1306, by Thermo Fisher Scientific (1 mentions)
Confocal laser scanning microscopy, by Leica (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (1 mentions)
Alexa fluor 647 conjugated goat anti mouse antibody, by Jackson ImmunoResearch (1 mentions)
Hoechst 33258, by Merck Group (1 mentions)
Eclipse ni, by Nikon (1 mentions)
Dako protein block, by Agilent Technologies (1 mentions)
Rabbit anti β catenin antibody, by Cell Signaling Technology (1 mentions)
Elyra s 1 lsm 880, by Zeiss (1 mentions)
Mouse anti n cadherin antibody, by BD (1 mentions)
Alexa fluor 568 goat anti mouse igg, by Thermo Fisher Scientific (1 mentions)
Prolong gold antifade reagent, by Thermo Fisher Scientific (1 mentions)
Saponin, by Merck Group (1 mentions)
4 protocols using mouse anti sarcomeric α actinin antibody
1
Whole-Mount Cardiac Tube Staining
2
Microgrooved Surfaces for Myogenic Differentiation
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C2C12
cells were seeded
onto the surfaces of microgrooved structures with different widths
at a concentration of 1 × 104 cells/mL and cultured
in growth medium for 3 days and myogenic differentiation medium for
5 days. Then, the cells were washed with PBS and fixed using 4% formaldehyde
for 15 min at room temperature. Next, the cells were immersed in 0.1%
Triton X-100 for 10 min and 1% bovine serum albumin (BSA) in 1x PBS
for 30 min at room temperature. The samples were then incubated with
1:200 mouse antisarcomeric α-actinin antibody (Sigma) in 1%
BSA at 4 °C overnight. Cells were washed 3 times with 1×
PBS before incubation with Alexa 488-conjugated goat antimouse antibody
at 1:400 in 1% BSA at room temperature in the dark for 60 min (Invitrogen).
Cell nuclei were stained with DAPI (Invitrogen) for 10 min at room
temperature in the dark. Finally, the cells were observed by confocal
laser scanning microscopy (Leica, Germany).
cells were seeded
onto the surfaces of microgrooved structures with different widths
at a concentration of 1 × 104 cells/mL and cultured
in growth medium for 3 days and myogenic differentiation medium for
5 days. Then, the cells were washed with PBS and fixed using 4% formaldehyde
for 15 min at room temperature. Next, the cells were immersed in 0.1%
Triton X-100 for 10 min and 1% bovine serum albumin (BSA) in 1x PBS
for 30 min at room temperature. The samples were then incubated with
1:200 mouse antisarcomeric α-actinin antibody (Sigma) in 1%
BSA at 4 °C overnight. Cells were washed 3 times with 1×
PBS before incubation with Alexa 488-conjugated goat antimouse antibody
at 1:400 in 1% BSA at room temperature in the dark for 60 min (Invitrogen).
Cell nuclei were stained with DAPI (Invitrogen) for 10 min at room
temperature in the dark. Finally, the cells were observed by confocal
laser scanning microscopy (Leica, Germany).
3
Immunofluorescent Labeling of Cardiac Tissue
4
Immunofluorescence Characterization of iPSC-CMs
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iPSC‐CMs cultured on 8‐chamber culture slides (ThermoFisher, 154461PK) were fixed with 100% methanol at −20°C for 10 min. Cells were blocked and permeabilized in Dako protein block (Agilent, X0909) containing 0.1% (w/v) saponin (Sigma‐Aldrich, 47036) at room temperature for 90 min. Cells were then incubated with primary antibodies (see below) diluted in the same blocking solution at 4°C for overnight. Then cells were washed with phosphate‐buffered saline (PBS) and incubated with secondary antibodies (see below) at room temperature for 1 h. Cells were washed with PBS and mounted with ProLong gold antifade reagent containing DAPI (ThermoFisher, P36931). Primary antibodies used were monoclonal rabbit anti‐Nav1.5 antibody targeting the C‐terminus (1:100, Cell Signaling, 14421), monoclonal mouse anti‐α‐sarcomeric actinin antibody (1:400, Sigma, A7811), rabbit anti‐β‐catenin antibody (1:100, Cell Signaling, 8480), and mouse anti‐N‐Cadherin antibody (BD Bioscience, 610920). Secondary antibodies used were goat anti‐mouse IgG Alexa Fluor‐568 (1:300, ThermoFisher, A‐11004) and anti‐rabbit IgG Alexa Fluor‐488 (1:300, ThermoFisher, A‐11008). Fluorescent cellular images were taken with a ZEISS confocal microscope equipped with the Airyscan technique for high‐resolution imaging (Zeiss Elyra S.1 LSM 880).
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