Fluorescent in situ hybridization was performed as described previously [48 (link)].
Briefly, testes were dissected in 1X PBS and then fixed in 4% formaldehyde/PBS for 45 minutes. After fixing, they were rinsed 2 times with 1X PBS, then resuspended in 70% EtOH, and left overnight at 4°C. The next day, testes were washed briefly in wash buffer (2X SSC and 10% deionized formamide), then incubated overnight at 37°C in the dark with 50 nM of Quasar 570 labeled Stellaris probe against dpp mRNA (LGC Biosearch Technologies, a gift from Michael Buszczak [49 (link)]) in the hybridization buffer containing 2X SSC, 10% dextran sulfate (Sigma-Aldrich Inc., St Louis, Missouri), 1 μg/μl of yeast tRNA (Sigma-Aldrich Inc.), 2 mM vanadyl ribonucleoside complex (NEB), 0.02% RNAse-free BSA (ThermoFisher), and 10% deionized formamide. On the third day, testes were washed 2 times for 30 minutes each at 37°C in the dark in the prewarmed wash buffer (2X SSC, 10% formamide) and then resuspended in a drop of VECTASHIELD with DAPI (Vector Lab, H-1200).
For quantification of the FISH signal, z-stacks were collected at 0.5 μm intervals using the same acquisition settings of for confocal microscopy using a Zeiss LSM800. The total number of particles in the hub was counted using Octane1.5.1 (Super-resolution Imaging and Single Molecule Tracking Software (https://github.com/jiyuuchc/Octane )).
Briefly, testes were dissected in 1X PBS and then fixed in 4% formaldehyde/PBS for 45 minutes. After fixing, they were rinsed 2 times with 1X PBS, then resuspended in 70% EtOH, and left overnight at 4°C. The next day, testes were washed briefly in wash buffer (2X SSC and 10% deionized formamide), then incubated overnight at 37°C in the dark with 50 nM of Quasar 570 labeled Stellaris probe against dpp mRNA (LGC Biosearch Technologies, a gift from Michael Buszczak [49 (link)]) in the hybridization buffer containing 2X SSC, 10% dextran sulfate (Sigma-Aldrich Inc., St Louis, Missouri), 1 μg/μl of yeast tRNA (Sigma-Aldrich Inc.), 2 mM vanadyl ribonucleoside complex (NEB), 0.02% RNAse-free BSA (ThermoFisher), and 10% deionized formamide. On the third day, testes were washed 2 times for 30 minutes each at 37°C in the dark in the prewarmed wash buffer (2X SSC, 10% formamide) and then resuspended in a drop of VECTASHIELD with DAPI (Vector Lab, H-1200).
For quantification of the FISH signal, z-stacks were collected at 0.5 μm intervals using the same acquisition settings of for confocal microscopy using a Zeiss LSM800. The total number of particles in the hub was counted using Octane1.5.1 (Super-resolution Imaging and Single Molecule Tracking Software (
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