After turning on the confocal microscope equipped with 40x and 63x oil immersion objectives, place the slide on the microscope stage. To image the wing disc associated muscle progenitors and indirect flight muscles or IFMs, set DAPI with an excitation of 405 and emission of 450 nanometers. Then select Alexa Fluor 488 with an excitation of 496 and emission of 519 nanometers, and Quasar 670 for the smFISH probes with an excitation of 647 and emission of 670 nanometers.
Locate the sample using a DAPI signal and UV lamp. Save the captured images as TIFF files. To image the adult muscle stem cells, set the excitation and emission wavelengths for DAPI and Alexa Fluor 488 as demonstrated previously.
Then set the wavelengths for Alexa Fluor 555 and Quasar 670 for the smFISH probes. Locate the sample and save the captured images as TIFF. Launch image J and navigate to the plugins menu.
Select Macros, then Edit to open the macros source code. To quantify Mef2 smFISH spots in larvae, set the log radius to three and log quality to 20. Then segment Mef2 positive nuclei with a blur of two, a nucleus scale parameter of 30, nucleus threshold of minus eight, and nucleus size of 300.
Initiate the macro by executing the run command. The macro will automatically load all images from the designated folder and quantify them sequentially. In muscle fibers, set the log radius to 2.5, log quality to 60, blur to two, nucleus scale parameter to 100, nucleus threshold to zero, and nucleus size to 300.
Initiate the macro by executing the run command. The macro will automatically load all images from the designated folder and quantify them sequentially. After analysis, check the results displayed in file FISH results and file nuclei results.
The Mef2 and Zfh1 transcripts were uniformly detected in the AMP population and colocalized with Mef2 and Zfh1 proteins respectively. A higher magnification of AMPs distinguished between transcription sites foci and mature mRNA scattered in the cytoplasm. Similarly, the transcription site and distribution of Mef2 and Zfh1 mRNA were examined in differentiated adult IFMs and associated stem cells.
The in-house built image J macro effectively detected and segmented Mef2 spots and muscle nuclei. The quantitative analysis of Mef2 mRNA per nuclei in adult IFMs and the AMPs were not significantly different. The quantification of Mef2 spot distribution showed that 92%of Mef2 mRNA is present in the cytoplasm, and 8%are associated with muscle nuclei.
