Neoblasts in G2/M phase (X1), or G0/G1 phase (X2), and differentiated cells (Xins) were isolated by Fluorescence-Activated Cell Sorting based on DNA content (Hoechst fluorescence) as reported by (23 (link)), following the procedures described previously (13 (link)).
For staining of the isolated cells, cell suspensions isolated by FACS were collected in CMFB and centrifuged at ∼300 g for 5 min at 4°C. Cells were washed in CMF, spotted onto poly-d -lysine coated coverslips (BD Biosciences), allowed to settle for ∼30 min, and fixed in 4% PFA (in PBS) for 20 min at room temperature. For SYTO RNAselect staining, fixation instead was performed for 10 min at −20°C in ice-cold methanol. Controls and treatment were always spotted on the same cover slip, and went through all staining steps using the same solutions in the same well. IF and FISH labelings were carried out similarly to the whole-mount protocol, with wash steps and antibody incubations shortened to 10 min and 1 hour, respectively.
For staining of the isolated cells, cell suspensions isolated by FACS were collected in CMFB and centrifuged at ∼300 g for 5 min at 4°C. Cells were washed in CMF, spotted onto poly-
