Immunofluorescence analysis (IFA) was as described (43 (link)). Antibodies for IFA were used at the following dilutions: mouse anti-HA-epitope IgG (Santa Cruz Biotechnology Inc.) at 1:1000, rabbit anti-Rab11 at 1:200 (54 (link)), rabbit anti-Rab5a at 1:200 (55 (link)), mouse anti-p67 at 1:1000 (56 (link)), mouse anti-BiP at 1:10 000 (57 (link)), rabbit anti-VSG221 at 1:1000 (55 (link)), rabbit anti-RabX2 at 1:50 (58 (link)), rabbit anti-IGP48 (this study) at 1:50. Secondary antibodies were used at the following dilutions: anti-mouse Oregon Green (Molecular Probes) at 1:1000 and anti-rabbit Cy3 (Sigma) at 1:1000. Cells were examined on a Nikon Eclipse E600 epifluorescence microscope fitted with optically matched filter blocks and a Hamamatsu ORCA charge-coupled-device camera. Digital images were captured using Metamorph software (Universal Imaging Corp.) on a computer running the Windows XP operating system (Microsoft Inc.) and the raw images processed using Photoshop CS6 software (Adobe Systems Inc.). Confocal z-sections were acquired using a Leica DMIRE2 microscope and deconvolved using Huygens Professional software.
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Immunofluorescence Analysis of Cellular Compartments
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mouse anti-HA-epitope IgG
anti-mouse Oregon Green
anti-rabbit Cy3
ORCA charge-coupled-device camera
Metamorph software
DMIRE2 microscope
Anti igg
Antibodies
Cells
Device
Dilutions
E600
Epitope
Immunofluorescence
Microscope
Molecular probes
Mouse
Orca
Rabbit
Corresponding Organization :
Other organizations : University of Dundee, University of Aberdeen
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- Antibody dilutions: mouse anti-HA-epitope IgG (1:1000), rabbit anti-Rab11 (1:200), rabbit anti-Rab5a (1:200), mouse anti-p67 (1:1000), mouse anti-BiP (1:10000), rabbit anti-VSG221 (1:1000), rabbit anti-RabX2 (1:50), rabbit anti-IGP48 (1:50)
- Secondary antibody dilutions: anti-mouse Oregon Green (1:1000), anti-rabbit Cy3 (1:1000)
- Immunofluorescence analysis (IFA) of the localization of HA-epitope, Rab11, Rab5a, p67, BiP, VSG221, RabX2, and IGP48 proteins
- Immunofluorescence analysis (IFA) protocol as described in the referenced literature (43)
- Microscopy setup: Nikon Eclipse E600 epifluorescence microscope with optically matched filter blocks, Hamamatsu ORCA charge-coupled-device camera, and Metamorph software for image capture and processing
- Confocal z-sections were acquired using a Leica DMIRE2 microscope and deconvolved using Huygens Professional software
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