Fluorescence Microscopy Colocalization Protocol

For fluorescence microscopy, cells were grown on coverslips and fixed for 20 min in 2% paraformaldehyde, quenched with 3% BSA, permeabilized with 0.1% saponin (Sigma-Aldrich), and incubated serially with the indicated primary and corresponding secondary antibodies. Images were taken with an Axiovert 200 M/ApoTome microscope and a confocal Exciter laser scanning microscope (Zeiss)^{30 (link)}. Colocalization was measured using AxioVision colocalization and Zen 2009 software (Zeiss). Pearson coefficients were calculated using the CoLocalizer Express software (CoLocalization Research Software).

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Radomski N., Franzke K., Matthiesen S., Karger A, & Knittler M.R. (2019). NK Cell-Mediated Processing Of Chlamydia psittaci Drives Potent Anti-Bacterial Th1 Immunity. Scientific Reports, 9, 4799.

Publication 2019

saponin Axiovert 200 M/ApoTome microscope Exciter laser scanning microscope Zen 2009 software

Antibodies Cells Confocal laser scanning microscope Fluorescence microscopy M 200 Microscope Paraformaldehyde Saponin

Corresponding Organization :

Other organizations : Friedrich-Loeffler-Institut

Top 5 similar protocols

Variable analysis

independent variables

Primary antibodies
Secondary antibodies

dependent variables

Colocalization between proteins
Pearson coefficients

control variables

Cells grown on coverslips
Cells fixed for 20 min in 2% paraformaldehyde
Cells quenched with 3% BSA
Cells permeabilized with 0.1% saponin (Sigma-Aldrich)
Microscopes used: Axiovert 200 M/ApoTome, confocal Exciter laser scanning (Zeiss)
Software used: AxioVision colocalization, Zen 2009 (Zeiss), CoLocalizer Express

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