Immunocytochemical Analysis of Cellular Processes

Immunostaining was performed as described^{17 (link)}. Cells were fixed with 4% paraformaldehyde for 30 min and permeabilised in phosphate-buffered saline containing 0.4% saponin, 1% bovine serum albumin and 2% normal goat serum at room temperature for 15 min. Cells were incubated with primary antibodies at 4 °C overnight and then with fluorescent dye-conjugated secondary antibodies (Life Technologies) at room temperature for 1 h. F-actin was visualised with Alexa647-conjugated phalloidin (Life Technologies)^{17 (link),30}. Endocytosis and cholesterol localisation were examined using FITC-dextran (Life Technologies)^{46 (link)} and filipin (SIGMA)^{47 (link)}, respectively. Fluorescence images were acquired with FV-1000 Confocal Microscope (OLYMPUS).

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Matsumoto N., Sekiya M., Tohyama K., Ishiyama-Matsuura E., Sun-Wada G.H., Wada Y., Futai M, & Nakanishi-Matsui M. (2018). Essential Role of the a3 Isoform of V-ATPase in Secretory Lysosome Trafficking via Rab7 Recruitment. Scientific Reports, 8, 6701.

Publication 2018

fluorescent dye-conjugated secondary antibodies Alexa647-conjugated phalloidin FITC-dextran filipin FV-1000 Confocal Microscope

Alexa647 Antibodies Bovine serum albumin Cells Cholesterol Confocal microscope Endocytosis F actin Filipin Fitc dextran Fluorescence Fluorescent dye Goat Paraformaldehyde Phalloidin Phosphate Saline Saponin Serum

Corresponding Organization :

Other organizations : Iwate Medical University, Doshisha Women's College of Liberal Arts, Osaka University

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Variable analysis

independent variables

Immunostaining protocol
Fixation with 4% paraformaldehyde
Permeabilisation with phosphate-buffered saline containing 0.4% saponin, 1% bovine serum albumin and 2% normal goat serum
Incubation with primary antibodies
Incubation with fluorescent dye-conjugated secondary antibodies
Visualization of F-actin with Alexa647-conjugated phalloidin
Examination of endocytosis using FITC-dextran
Examination of cholesterol localisation using filipin

dependent variables

Immunostaining pattern
Endocytosis
Cholesterol localisation

control variables

Incubation time for primary antibodies (overnight at 4 °C)
Incubation time for secondary antibodies (1 h at room temperature)

positive controls

Not explicitly mentioned

negative controls

Not explicitly mentioned

Annotations

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