Tissue freezing medium

Submandibular and sublingual salivary glands were removed together, weighed, and fixed in 4% paraformaldehyde (PFA) in 1X phosphate buffered saline (1X PBS) for 2 h at 4°C. Glands were washed in three consecutive 1X PBS washes, and incubated in an increasing sucrose gradient progressing from 5%, to 10%, to 15% sucrose for 1 h each and then transferred to 30% sucrose overnight at 4°C. After overnight incubation, glands were transferred to 15% sucrose, 50% tissue freezing media (Tissue Freezing Medium, Electron Microscopy Sciences) overnight at 4°C. Glands were transferred to Tissue Freezing Medium and then frozen over liquid nitrogen. Ten-micron (10 µm) sections were collected on Superfrost Plus glass slides (Electron Microscopy Sciences) using a Leica CM 1860 cryostat. Slides were collected by taking three consecutive 10 µm sections per slide until the entire tissue was sectioned. Sections were dried for 30 min at room temperature and stored at −80°C. Slides were returned to room temperature before staining.

One anemone from each of the eight 500-ml tanks was collected at 11:00 a.m. (~6 hours into the light period), immediately snap-frozen in liquid nitrogen, embedded in the Tissue Freezing Medium (Electron Microscopy Sciences), and stored at −80°C until cryosectioning. The cryostat (CM3050 S, Leica) was prechilled to a chamber temperature of −23°C, and samples were equilibrated to the chamber temperature for 20 min and then sectioned at a thickness of 8 μm. Sections were then transferred onto precooled ribonuclease (RNase)–free polyester membrane metal frame slides (Leica) and air-dried for 5 min in a RNase-free fume hood.
Gastrodermal and epidermal cell layers were identified in sections (mostly from tentacles) at both ×10 and ×20 magnifications using a Leica LMD 6000 microscope with Leica filter cubes B/G/R and A. Each region of interest was traced individually using the LMD software and dissected using an ultraviolet laser beam. The dissected pieces were collected in CapSure Macro LCM Caps (Thermo Fisher Scientific) containing 40 μl of RNA extraction buffer from the Arcturus PicoPure RNA Isolation Kit (Thermo Fisher Scientific). The harvested cells were lysed by incubation at 42°C for 30 min, vortexed briefly, and then kept at −80°C until further processing.

Immediately after isolation, whole hearts were perfused with RNase-free PBS to flush out blood (~1 min) and then 4% paraformaldehyde (Electron Microscopy Sciences) in RNase-free PBS until the heart was stiffened to the extent that it could no longer perfuse (~15–20 min). The heart was then left in 50 mL 4% paraformaldehyde in RNase-free PBS in a conical tube rotating at 4 ˚C overnight. Next, at room temperature, the heart was washed three times for 30 min in RNase-free PBS and then placed in 50 mL 15% sucrose solution (Thermo Fisher) rotating at 4 ˚C overnight, or until the heart no longer floated. Finally, the heart was placed in 50 mL 30% sucrose solution rotating at 4 ˚C overnight, or until the heart no longer floated. Hearts were then embedded in tissue freezing medium (Electron Microscopy Sciences) in plastic mold using liquid nitrogen-cooled isopentane prior to sectioning. Frozen blocks were sectioned to a thickness of ≤10 µm and stored at −80 ˚C.