Primary liver cells fixed in 4 methanol-free % PFA for 5 minutes were blocked and stained in IF buffer (1x PBS, 10 mg/ml BSA, 0.02% SDS, 0.1% Triton X-100). Primary and secondary antibodies was incubated for 1 hour at RT or overnight at 4°C with IF buffer washes between and after incubations. Cells were labeled with Hoechst 33342 (Life Technologies) for 5 minutes before mounting using VECTASHIELD (Vector Laboratories). Antibodies used: mAb414 (Covance or BioLegend); anti-Nup210 (Bethyl Laboratories, Inc.); anti-Pom121 (ThermoFisher Scientific); anti-Nup88 (22, BD Transduction Laboratories); anti-Nup107 (a kind gift from Martin Hetzer9 (link), The Salk Institute, La Jolla CA, USA); anti-Nup98 (39A3, Cell Signaling Technology); anti-Nup93 (F2, Santa Cruz BioTechnology); anti-Lamin A (Sigma Aldrich); anti-Lamin B1 (Abcam); anti-Cav1 (Cell Signaling Technology), and anti-Cav2 (65, BD Biosciences). Images were taken using a Leica SP8 confocal microscope and analyzed using the Leica Application Suite X software v3.1.5.16308, ImageJ v2.0.0-rc-54/1.51h (NIH), and Adobe Photoshop CS5.1 v12.1 x64.
Protocol detail
Immunofluorescence Staining of Primary Liver Cells
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Hoechst 33342
VECTASHIELD
mAb414
anti-Nup210
anti-Pom121
anti-Nup88
anti-Nup107
anti-Nup98
anti-Nup93 (F2,
anti-Lamin A
anti-Lamin B1
anti-Cav1
anti-Cav2
SP8 confocal microscope
Anti b1
Antibodies
Buffer
Cav1
Cav2
Cells
Confocal microscope
Hoechst 33342
Lamin
Lamin a
Lamin b1
Liver cells
Methanol
Nup93
Nup98
Triton x 100
Vector
Corresponding Organization :
Other organizations : Discovery Institute, Sanford Burnham Prebys Medical Discovery Institute
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Variable analysis
independent variables
- Primary liver cells
- Fixation with 4% methanol-free PFA for 5 minutes
- Cellular localization/distribution of proteins (mAb414, Nup210, Pom121, Nup88, Nup107, Nup98, Nup93, Lamin A, Lamin B1, Cav1, Cav2)
- Blocking and staining in IF buffer (1x PBS, 10 mg/ml BSA, 0.02% SDS, 0.1% Triton X-100)
- Primary and secondary antibody incubation time (1 hour at RT or overnight at 4°C)
- IF buffer washes between and after incubations
- Labeling with Hoechst 33342 for 5 minutes before mounting
- No positive controls specified
- No negative controls specified
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