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Spinning disc confocal microscopy

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Spinning disc confocal microscopy is an imaging technique that uses a rotating disc with pinholes to rapidly scan a sample and produce high-resolution, real-time images. The core function of this technology is to provide optical sectioning and improved image contrast by rejecting out-of-focus light, enabling the visualization of samples with increased clarity and detail.

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Lab products found in correlation

Vectashield, by Vector Laboratories (3 mentions) Cy3 conjugated goat anti mouse antibody, by Jackson ImmunoResearch (2 mentions) Alexa 488 conjugated donkey anti rabbit, by Jackson ImmunoResearch (2 mentions) Anti flag, by Cell Signaling Technology (1 mentions)

3 protocols using spinning disc confocal microscopy

1

Xenopus Oocyte Immunofluorescence Assay

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Whole-mount immunofluorescence assays using Xenopus oocytes were performed as described (Zheng et al., 2017 ). Briefly, oocytes were washed in PBS, fixed in 4% paraformaldehyde for 15 min, washed three times in PBS plus 50 mM NH4Cl, and then permeabilized with 0.1% Triton X-100 for 4 min. Oocytes werethen blocked in PBS plus 3% skim milk for 30 min and then incubated overnight with indicated primary antibodies, followed by incubation with secondary Alexa-488-conjugated donkey anti-rabbit or Cy3-conjugated goat anti-mouse antibodies (Jackson ImmunoResearch Laboratories, West Grove, PA) for 30 min. Oocytes were then mounted in Vectashield (Vector Labs, Burlington, ON) and examined on an AIVI spinning disc confocal microscopy (Cell Imaging Facility, Faculty of Medicine and Dentistry, University of Alberta).
Zheng W., Cai R., Hofmann L., Nesin V., Hu Q., Long W., Fatehi M., Liu X., Hussein S., Kong T., Li J., Light P.E., Tang J., Flockerzi V., Tsiokas L, & Chen X.Z. (2018). Direct Binding between Pre-S1 and TRP-like Domains in TRPP Channels Mediates Gating and Functional Regulation by PIP2. Cell reports, 22(6), 1560-1573.
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2

Immunofluorescence Analysis of Oocyte Proteins

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Whole mount immunofluorescence experiments in oocytes were carried out as previous63 (link). Briefly, oocytes were fixed in 4% paraformaldehyde for 15 min, followed by permeabilization using 0.1% Triton X-100 for 4 min. Oocytes were blocked in 3% skim milk at RT for 30 min, followed by overnight incubation at 4 °C with anti-Flag (#14793, 1:200) or -HA (#3724, 1:200) primary antibodies from Cell Signaling Technology (Danvers, MA), and then incubated with a secondary donkey anti-rabbit lgG conjugated with AlexaFluor 488 (Jackson ImmunoResearch Laboratories, West Grove, PA) for 30 min at RT. Oocytes were mounted in Vectashield (Vector Labs, Burlington, ON) for fluorescence examination using an AIVI spinning disc confocal microscopy (Cell Imaging Facility, Faculty of Medicine and Dentistry, University of Alberta).
Zheng W., Yang X., Hu R., Cai R., Hofmann L., Wang Z., Hu Q., Liu X., Bulkley D., Yu Y., Tang J., Flockerzi V., Cao Y., Cao E, & Chen X.Z. (2018). Hydrophobic pore gates regulate ion permeation in polycystic kidney disease 2 and 2L1 channels. Nature Communications, 9, 2302.
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3

Xenopus Oocyte Immunofluorescence Assay

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Whole-mount immunofluorescence assays using Xenopus oocytes were performed as described (Zheng et al., 2017 ). Briefly, oocytes were washed in PBS, fixed in 4% paraformaldehyde for 15 min, washed three times in PBS plus 50 mM NH4Cl, and then permeabilized with 0.1% Triton X-100 for 4 min. Oocytes werethen blocked in PBS plus 3% skim milk for 30 min and then incubated overnight with indicated primary antibodies, followed by incubation with secondary Alexa-488-conjugated donkey anti-rabbit or Cy3-conjugated goat anti-mouse antibodies (Jackson ImmunoResearch Laboratories, West Grove, PA) for 30 min. Oocytes were then mounted in Vectashield (Vector Labs, Burlington, ON) and examined on an AIVI spinning disc confocal microscopy (Cell Imaging Facility, Faculty of Medicine and Dentistry, University of Alberta).
Zheng W., Cai R., Hofmann L., Nesin V., Hu Q., Long W., Fatehi M., Liu X., Hussein S., Kong T., Li J., Light P.E., Tang J., Flockerzi V., Tsiokas L, & Chen X.Z. (2018). Direct Binding between Pre-S1 and TRP-like Domains in TRPP Channels Mediates Gating and Functional Regulation by PIP2. Cell reports, 22(6), 1560-1573.
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