Human PASMCs were fixed in 4% paraformaldehyde for 15 min and permeabilized with 0.1% Triton X-100 in PBS for 10 min at room temperature. The cells were blocked with 5% BSA (Sigma) for 1 h at room temperature, washed with PBS, labeled with anti-Ki67 antibody (Sigma) or anti-ATF6 antibody (Santa Cruz Biotechnology) for 2 h at room temperature, rinsed in PBS, and incubated with the AlexaFluor-488 antibody (ZSGB-BIO) for 60 min at room temperature. The nuclei were stained with DAPI and fluorescent staining was visualized using a BX-61 microscope (Olympus) or confocal microscope (Leica TCS SP8), as previously described 23 (link).
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Immunofluorescence Staining of Human PASMCs
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Anti antibody
Antibody
Atf6
Cells
Confocal microscope
Dapi
Human
Microscope
Nuclei
Paraformaldehyde
Triton x 100
Corresponding Organization : Peking Union Medical College Hospital
Other organizations : Capital Medical University, Beijing Children’s Hospital
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Variable analysis
independent variables
- Paraformaldehyde concentration (4%)
- Permeabilization with Triton X-100 (0.1% in PBS)
- Blocking with BSA (5%)
- Incubation with primary antibodies (anti-Ki67 or anti-ATF6)
- Incubation with secondary antibody (AlexaFluor-488)
- Expression of Ki67 or ATF6 proteins
- Fluorescent staining visualized using microscopy
- Incubation time for fixation (15 min)
- Incubation time for permeabilization (10 min)
- Incubation time for blocking (1 h)
- Incubation time for primary antibody (2 h)
- Incubation time for secondary antibody (60 min)
- Nuclear staining with DAPI
- Positive control: Not explicitly mentioned
- Negative control: Not explicitly mentioned
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