We used Phalloidin-iFluor647 to stain Actin in HaCaT cells in STED microscopy. The reagent yielded very bright samples and the iFluor reagent worked quite nicely with STED. There was a loss in fluorescence intensity of about 60-70% compared to confocal image, but that should not be a problem in well stained samples.
The cells were fixed with 4% PFA and permeabilized with 0,2% Triton X-100. Samples were blocked with 3% BSA/PBS for 30 min. We diluted the reagent stock 1:1000 accordingly to the manufacturers instructions and added the reagent during the secondary antibody step (dilution in 3% BSA/PBS). The incubation time was 1 h at room temperature.
The cells were fixed with 4% PFA and permeabilized with 0,2% Triton X-100. Samples were blocked with 3% BSA/PBS for 30 min. We diluted the reagent stock 1:1000 accordingly to the manufacturers instructions and added the reagent during the secondary antibody step (dilution in 3% BSA/PBS). The incubation time was 1 h at room temperature.
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MR. Membrane Biochemistry
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Submitted Dec 04 2017