Fluorescence Imaging of PEX Proteins

Cells expressing PEX26 or PEX15 constructs were analyzed by direct fluorescence and immunofluorescence experiments. If not indicated otherwise, cells were transfected for 48 hours using Effectene (Qiagen). Fixation, permeabilization, incubation with antibodies, and mounting was carried out as described58 (link)59 (link). The following antibodies were used: anti-PEX14 (ProteinTech, rabbit polyclonal, dilution 1:200) and anti-Myc (Cell Signaling, mouse monoclonal, dilution 1:2000), anti-Catalase (Oxis International, rabbit polyclonal, 1:1000), anti-mouse Alexa488 (MoBiTech, dilution 1:200), and anti-rabbit Cy3 (Jackson ImmunoResearch, dilution 1:200). Yeast cells were mounted in low-melting agarose for imaging. Samples were analyzed using a 100x oil immersion objective (NA 1.3) on a Zeiss Imager M1 fluorescence wide field microscope with a HRm Camera and the Axiovision 4.8 acquisition software. For yeast microscopy widefield images were contrast-enhanced and false-colored in blue to show cell contours.

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Buentzel J., Vilardi F., Lotz-Havla A., Gärtner J, & Thoms S. (2015). Conserved targeting information in mammalian and fungal peroxisomal tail-anchored proteins. Scientific Reports, 5, 17420.

Publication 2015

Effectene anti-Myc anti-rabbit Cy3 Imager M1 Axiovision 4.8

Agarose Antibodies Catalase Cell Dilution Effectene Fluorescence Fluorescence microscope Immersion Immunofluorescence Microscopy Mouse Oxis Rabbit Yeast

Corresponding Organization :

Other organizations : University of Göttingen

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Variable analysis

independent variables

Cells expressing PEX26 or PEX15 constructs

dependent variables

Localization and expression of PEX14, Myc, and Catalase proteins as analyzed by direct fluorescence and immunofluorescence experiments

control variables

Fixation, permeabilization, incubation with antibodies, and mounting procedures as described in the referenced publications
Yeast cells were mounted in low-melting agarose for imaging

controls

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

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