Cells were washed with DPBS and fixed in 10 % formalin solution (HT501128, Sigma-Aldrich) for 15 min followed by permeabilization with 50 μg/mL Digitonin (D141, Sigma-Aldrich) for 10 min at RT. Cells were blocked in blocking solution (1 % Bovine Serum Albumin (A7030, Sigma-Aldrich), 5 % Donkey Serum (D9663, Sigma-Aldrich) and 5 % Goat Serum (31873, ThermoFisher Scientific) in DPBS) for 30 min at RT. Cells were then incubated with 1 % BSA solution containing primary antibodies at various dilutions (Table 2 ) overnight at 4 °C followed by 1 % BSA solution containing secondary antibodies at various dilutions (Table 2 ) for 30 min at RT. Nuclei were counterstained with the Molecular Probes NucBlue Fixed Cell ReadyProbes Reagent (R37606, ThermoFisher Scientific). Cells were imaged using the ECHO Revolve microscope.
Molecular probes nucblue fixed cell readyprobes reagent
Manufactured by Thermo Fisher Scientific
The Molecular Probes NucBlue Fixed Cell ReadyProbes Reagent is a fluorescent dye that binds to DNA, allowing for the visualization of cell nuclei in fixed and permeabilized cells. The reagent provides a simple and convenient way to stain cell nuclei for various microscopy and flow cytometry applications.
Automatically generated - may contain errors
Lab products found in correlation
Digitonin, by Merck Group (5 mentions)
Donkey serum, by Merck Group (4 mentions)
Ht501128, by Merck Group (4 mentions)
Bovine serum albumin (bsa), by Merck Group (3 mentions)
Goat serum, by Thermo Fisher Scientific (2 mentions)
Paraformaldehyde solution, by Merck Group (2 mentions)
D9663, by Merck Group (2 mentions)
7 protocols using molecular probes nucblue fixed cell readyprobes reagent
1
Immunofluorescence Staining of Cultured Cells
2
Immunocytochemistry of iPSCs
3
Immunocytochemistry Protocol for Cell Quantification
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Cells (passages greater than 20) were fixed in 4 % paraformaldehyde (PFA) solution (Sigma-Aldrich #HT501128) for 20 min and permeabilized with 50 μg/mL digitonin (Sigma-Aldrich #D141) for 10 min at room temperature (RT). The cells were blocked for 30 min at RT in blocking solution (DPBS with 1 % Bovine Serum Albumin and 5 % serum (Donkey Serum, Sigma-Aldrich #D9663)) and then incubated overnight at 4 °C with primary antibodies diluted in blocking solution. After aspirating the primary antibodies, the cells were incubated for 30 min at RT with secondary antibodies diluted in blocking solution (Table 2 ). The quantification of positive nuclei was performed by counting the number of positive cells in multiple fields of view for each biological replicate. The standard deviation was calculated based on the variability observed among multiple biological replicates. Image analysis was conducted using ImageJ software (Fig. 1B ), and a one-way ANOVA with a Dunnett-test for multiple comparisons was used to compare the percentage of positive cells in different conditions. Nuclear staining was performed using the Molecular Probes NucBlue Fixed Cell Ready Probes Reagent (Thermo Fisher Scientific #R37606) (Fig. 1C & E ).
4
Immunofluorescence Staining of Cells
5
Immunofluorescence Staining of iPSCs
6
Immunofluorescence Staining of Cells
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Cells were fixed in 10% formalin solution (Sigma-Aldrich #HT501128) for 15 min and permeabilized with 50 μg/mL digitonin (Sigma-Aldrich #D141) for 10 min at room temperature (RT), then blocked for 30 min at RT in blocking solution (DPBS with 1% Bovine Serum Albumin and 5% serum (Donkey Serum, Sigma-Aldrich #D9663)). Cells were incubated overnight at 4 °C with primary antibodies diluted in blocking solution. Cells were incubated for 30 min at RT with secondary antibodies diluted in blocking soliution (Table 2 ). Nuclei were stained with the Molecular Probes NucBlue Fixed Cell ReadyProbes Reagent (ThermoFisher Scientific #R37606).
7
Immunofluorescence Staining of Cultured Cells
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