Immunofluorescence Microscopy for Protein Localization

Cells were fixed at room temperature (RT) in 3.8% (vol/vol) formaldehyde in phosphate buffered saline (PBS) and permeabilized in PBS containing 0.1% (vol/vol) saponin (Sigma-Aldrich) or 0.1% (vol/vol) Triton X-100 (Sigma-Aldrich). Coverslips were labeled with primary and secondary antibodies as previously described (Connell et al., 2009 (link)). Slides were analyzed with an LSM880 confocal microscope (100 × NA 1.40 oil immersion objective, 37°C), LSM780 confocal microscope (63 × NA 1.40 oil immersion objective, 37°C), or AxioImager Z2 Motorized Upright Microscope (63 × NA 1.40 oil immersion objective, RT, Axiocam 506; ZEISS), all with ZEN analysis software (ZEISS). Images were subsequently processed using ImageJ, Adobe Photoshop, Adobe Illustrator, and ZEN analysis software. Co-localization of proteins was quantified using Volocity (PerkinElmer).

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Allison R., Edgar J.R, & Reid E. (2019). Spastin MIT Domain Disease-Associated Mutations Disrupt Lysosomal Function. Frontiers in Neuroscience, 13, 1179.

Publication 2019

saponin Triton X-100 LSM880 confocal microscope AxioImager Z2 Motorized Upright Microscope Axiocam 506 Volocity

Antibodies Cells Confocal microscope Formaldehyde Immersion Microscope Phosphate Proteins Saline Saponin Triton x 100

Corresponding Organization : University of Cambridge

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Variable analysis

independent variables

Permeabilization method (0.1% saponin or 0.1% Triton X-100)

dependent variables

Localization and co-localization of proteins (analyzed using LSM880 confocal microscope, LSM780 confocal microscope, or AxioImager Z2 Motorized Upright Microscope)

control variables

Formaldehyde fixation at room temperature (RT)
Phosphate buffered saline (PBS)
Primary and secondary antibody labeling (as previously described in Connell et al., 2009)
Temperature during microscopy (37°C for confocal microscopes, RT for AxioImager Z2 Motorized Upright Microscope)

controls

Positive control: Not explicitly mentioned
Negative control: Not explicitly mentioned

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