Fixation and Immunostaining of Cells
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Corresponding Organization :
Other organizations : University of Cambridge, The Honourable Society of Lincoln's Inn, Cancer Research UK
Protocol cited in 28 other protocols
Variable analysis
- Fixation time (15 min)
- Fixation temperature (20-24 °C)
- Immunofluorescence signal
- Fixative concentrations (4% formaldehyde, 0.2% glutaraldehyde)
- Rinsing steps (3 times with PBS containing 50 mM NH4Cl)
- Blocking and permeabilization (45 min with buffer A containing 5% NGS, 50 mM NH4Cl, 0.5% saponin)
- Primary antibody incubation (1 h in buffer A with 5% NGS, 0.1% saponin)
- Secondary antibody incubation (45 min in buffer A with 5% NGS, 0.1% saponin)
- Post-fixation (2% formaldehyde in PBS for 10 min on ice)
- Mounting (ProLong Gold with DAPI)
- 100 nM GST–PH-PLCδ1 as a probe for PtdIns(4,5)P2
- Removal of GST-tagged protein by two rinses with buffer A
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