Tfm blocks

Planarians were killed in chilled 2% HCl in PBS (RNAse-Free) and then fixed in 100% Acetone for 1 hour at −20°C. Planarians were then incubated in 10, 20, 30% sucrose in PBS (RNAse-Free) for 20 minutes each before embedding in Tissue Freezing Medium (TFM) blocks (Ted Pella). The samples were then cryosectioned at 16 mm thickness onto PEN membrane slides (Thermo Fisher). The slides were stained with 1% cresyl violet and 1% eosin Y (Sigma) in 75% ethanol (Clément-Ziza et al., 2008 (link)). The tissue samples were dissected using Veritas Laser Capture Microdissection System (Arcturus) and captured on to Capsure HS LCM Caps (Thermo Fisher). After capture, RNA was isolated using the Arcturus PicoPure RNA isolation kit (Thermo Fisher). Ovarian tissue samples were collected from 14 worms (10 worms used for cross-sections and 4 worms used for sagittal sections) for each replicate (3 replicates total). Testis- and non-gonadal-tissue samples were collected from 6 worms for each replicate (4 worms used for cross-sections and 2 worms used for sagittal sections). RNA concentrations were determined using Qubit Fluorometer (Thermo Fisher), and RNA integrity was analyzed using 2100 Bioanalyzer Instrument (Agilent).