Epifluorescence micrographs were obtained using an epifluorescence microscope (Nikon TiE) using a Cool Snap HQ2 or QuantEM digital camera. To be able to compare the images shown, fluorescence micrographs from the same wavelengths (Cy3 or Cy5) within an individual experiment were acquired with the same exposure time, and the display scales of the representative images from each condition were equalized. For mRNA distribution analysis, serial Z-sections (0.5-μm steps, between 5 and 7 μm total distances) were acquired, and a maximum projection image was generated using the Nikon Elements software. For quantification of the polarization and distribution of mRNAs, a manual mask was generated using ImageJ, and the dispersion and polarization indices were calculated using the script described (Park et al. 2012 (link)). Cells that contained bright STIC probe aggregates were not imaged for mRNA distribution analysis.
Protocol detail
Microscopic Imaging and Analysis of mRNA Distribution
Partial Protocol Preview
This section provides a glimpse into the protocol.
The remaining content is hidden due to licensing restrictions, but the full text is available at the following link:
Access Free Full Text.
Corresponding Organization :
Other organizations : Stony Brook University
Top 5 similar protocols
Variable analysis
independent variables
- Wavelength (Cy3 or Cy5)
- Exposure time for fluorescence micrographs
- MRNA distribution and polarization indices
- Presence of bright STIC probe aggregates in cells
- Epifluorescence microscope (Nikon TiE) used
- Digital camera (Cool Snap HQ2 or QuantEM) used
- Display scales of representative images equalized
- Total distance of Z-sections (5-7 μm)
- Cells containing bright STIC probe aggregates were not imaged for mRNA distribution analysis
Annotations
Based on most similar protocols
Etiam vel ipsum. Morbi facilisis vestibulum nisl. Praesent cursus laoreet felis. Integer adipiscing pretium orci. Nulla facilisi. Quisque posuere bibendum purus. Nulla quam mauris, cursus eget, convallis ac, molestie non, enim. Aliquam congue. Quisque sagittis nonummy sapien. Proin molestie sem vitae urna. Maecenas lorem.
Sign up for free or login to display all annotations
As authors may omit details in methods from publication, our AI will look for missing critical information across the 5 most similar protocols.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!