Confocal Imaging of Immunostained Cells

Immunostaining was performed as described previously^{6 (link),35 (link)}. Cells were fixed with 4% paraformaldehyde, permeabilized with P-sol (phosphate-buffered saline containing 0.4% saponin, 1% bovine serum albumin, and 2% normal goat serum), and incubated with primary antibodies in P-sol at 4 °C overnight, followed by fluorophore-conjugated secondary antibodies for 1 h at room temperature. Fluorescence images were acquired using an FV-1000 confocal microscope equipped with a 100 × objective, NA 1.40, and analyzed with FV10-ASW software (OLYMPUS).

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Matsumoto N., Sekiya M., Sun-Wada G.H., Wada Y, & Nakanishi-Matsui M. (2022). The lysosomal V-ATPase a3 subunit is involved in localization of Mon1-Ccz1, the GEF for Rab7, to secretory lysosomes in osteoclasts. Scientific Reports, 12, 8455.

Publication 2022

FV10-ASW software

Antibodies Bovine serum albumin Cells Confocal microscope Fluorescence Goat Paraformaldehyde 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

Primary antibodies
Fluorophore-conjugated secondary antibodies

dependent variables

Fluorescence images

control variables

4% paraformaldehyde for cell fixation
P-sol (phosphate-buffered saline containing 0.4% saponin, 1% bovine serum albumin, and 2% normal goat serum) for permeabilization and antibody incubation
FV-1000 confocal microscope with 100 × objective, NA 1.40, and FV10-ASW software (OLYMPUS) for image acquisition and analysis

controls

Positive control: Not specified
Negative control: Not specified

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