Whole-mount in situ hybridization (WISH) was performed as previously described [31 (link)]. The primers used to amplify for probe synthesis are listed in Table A in S1 Table . Then PCR products were used to generate probes using the DIG RNA labeling Kit (Roche Applied Science, Penzberg, Germany). The WISH images were captured using the SteREO Discovery V20 microscope (Carl Zeiss, Germany).
Whole-mount antibody staining was performed as previously described [51 (link)]. Primary antibodies anti-pRS6 (S240/244) (1:500; #2215 Cell Signaling, USA), anti-LC3B (1:1000; ab192890, Abcam), anti-Dendra2 (1:1000, AB821, Evrogen, Moscow, Russia). Secondary antibodies used in the study are donkey anti-goat IgG Alexa fluor 568-conjugated (1:1000, Invitrogen) and donkey anti-rabbit IgG Alexa fluor 488-conjugated (1:1000, Invitrogen). Images were captured using ZEN2010 software equipped on an LSM880 confocal microscope (Carl Zeiss).
Whole-mount antibody staining was performed as previously described [51 (link)]. Primary antibodies anti-pRS6 (S240/244) (1:500; #2215 Cell Signaling, USA), anti-LC3B (1:1000; ab192890, Abcam), anti-Dendra2 (1:1000, AB821, Evrogen, Moscow, Russia). Secondary antibodies used in the study are donkey anti-goat IgG Alexa fluor 568-conjugated (1:1000, Invitrogen) and donkey anti-rabbit IgG Alexa fluor 488-conjugated (1:1000, Invitrogen). Images were captured using ZEN2010 software equipped on an LSM880 confocal microscope (Carl Zeiss).
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