Anti mouse alexafluor 647

INS-1E cells expressing ER-Catalase N244 or WT catalase without an ER-specific targeting signal were seeded at a density of 100 000 cells/well on LabTek chamber slides (Nunc, Roskilde, Denmark). After 48 h, the cells were washed twice with PBS and subsequently fixed with 4% paraformaldehyde at room temperature for 1 h. After washing, the cells were permeabilised and blocked with PBS containing 0.2% Triton X-100 and 1% BSA. The cells were incubated with primary antibodies (anti-PDI, ab5484, Abcam, Cambridge, UK, 1:100 dilution; anticatalase, 100-4151, Rockland Immunochemicals, Inc., Limerick, PA, USA, 1:500 dilution) diluted in PBS containing 0.1% Triton X-100 and 0.1% BSA at room temperature for 1 h. Thereafter the cells were washed with PBS and incubated with specific secondary antibodies (anti-mouse-Alexa Fluor 647, or anti-rabbit-Alexa Fluor 488, Jackson ImmunoResearch Laboratories, West Grove, PA, USA; 1:200 dilutions) for 1 h in the dark. Afterwards the cells were washed and the nuclei were counterstained with 300 nM DAPI for 5 min at room temperature. Finally, the cells were washed and mounted with Mowiol/DABCO anti-photobleaching mounting media (Sigma). Stained cells were examined with an Olympus IX81 inverted microscope (Olympus) and microscopic images were post-processed using AutoDeblur and AutoVisualize (Autoquant Imaging, New York, NY, USA).