Cells were plated on glass coverslips and collected after 3 days of differentiation. The coverslips were fixed with 4% paraformaldehyde in PBS for 15 min, permeabilized in 0.5% Triton X-100 in PBS, and blocked in 5% goat serum. The coverslips were incubated with primary antibody MHC 22287–1-AP (Proteintech) overnight at 4°C and then with Alexa Fluor 555-conjugated secondary antibody (Thermo Fisher Scientific) for 1 h. Cells were stained with Hoechst 33342 (1 μg/mL; Invitrogen) for 2 min at room temperature, washed, and then mounted with ProLong Gold (Invitrogen). The primary and secondary antibodies were diluted 1:400 and 1:1000, respectively. Microscopy was performed using the Zeiss Axio Observer Live Cell microscope and ImageJ Software for analysis (Schneider et al., 2012 (link)).
Axio observer live cell microscope
Manufactured by Zeiss
The Axio Observer Live Cell microscope is a high-performance inverted microscope designed for live cell imaging. It features a motorized stage, autofocus, and environmental control to maintain optimal conditions for cell culture during long-term observation.
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3 protocols using axio observer live cell microscope
1
Immunofluorescence Assay for Myosin Heavy Chain
2
Immunofluorescence Assay for Myosin Heavy Chain
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Cells were plated on glass coverslips and collected after 3 days of differentiation. The coverslips were fixed with 4% paraformaldehyde in PBS for 15 min, permeabilized in 0.5% Triton X-100 in PBS, and blocked in 5% goat serum. The coverslips were incubated with primary antibody MHC 22287–1-AP (Proteintech) overnight at 4°C and then with Alexa Fluor 555-conjugated secondary antibody (Thermo Fisher Scientific) for 1 h. Cells were stained with Hoechst 33342 (1 μg/mL; Invitrogen) for 2 min at room temperature, washed, and then mounted with ProLong Gold (Invitrogen). The primary and secondary antibodies were diluted 1:400 and 1:1000, respectively. Microscopy was performed using the Zeiss Axio Observer Live Cell microscope and ImageJ Software for analysis (Schneider et al., 2012 (link)).
3
Immunofluorescence Staining of Differentiated Cells
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Cells were plated on glass coverslips and collected after 3 days of differentiation. The coverslips were fixed with 4% paraformaldehyde in PBS for 15 min, permeabilized in 0.5%
Triton X-100 in PBS, and blocked in 5% goat serum. The coverslips were incubated with primary antibody MHC 22287-1-AP (Proteintech) overnight at 4 °C and then with Alexa
Fluor 555-conjugated secondary antibody (Thermo Fisher Scientific) for 1 h. Cells were stained with Hoechst 33342 (1 μg/mL; Invitrogen) for 2 min at room temperature, washed, and then mounted with ProLong Gold (Invitrogen). The primary and secondary antibodies were diluted 1:400 and 1:1000, respectively. Microscopy was performed using the Zeiss Axio Observer Live Cell microscope and ImageJ Software for analysis (Schneider et al., 2012) .
Triton X-100 in PBS, and blocked in 5% goat serum. The coverslips were incubated with primary antibody MHC 22287-1-AP (Proteintech) overnight at 4 °C and then with Alexa
Fluor 555-conjugated secondary antibody (Thermo Fisher Scientific) for 1 h. Cells were stained with Hoechst 33342 (1 μg/mL; Invitrogen) for 2 min at room temperature, washed, and then mounted with ProLong Gold (Invitrogen). The primary and secondary antibodies were diluted 1:400 and 1:1000, respectively. Microscopy was performed using the Zeiss Axio Observer Live Cell microscope and ImageJ Software for analysis (Schneider et al., 2012) .
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