Cm1850 uv cryostat

Lewis lung carcinoma tumors were collected and fixed in ice cold 4% paraformaldehyde before being rinsed in PBS and cryo-protected overnight in 30% sucrose. Tissues were then embedded in O.C.T. Compound (Sakura, AJAlphen aan den Rijn, The Netherlands) and cut in a CM1850 UV cryostat (Leica Biosystems, Wetzlar, Germany). At least five cryosections (6 µm) were assayed for apoptosis by the TUNEL method (DeadEnd Fluorometric TUNEL System), according to the manufacturer’s protocol (46 (link), 47 (link)). Samples were counterstained with DAPI, mounted with Vectashield (Vector Laboratories, Burlingame, CA, USA), and examined using a DMI4000 B automated inverted microscope equipped with a DCF310 digital camera (Leica Microsystems, Wetzlar, Germany). Image acquisition was controlled by the Leica LAS AF software.

Eyes of wild-caught A. anableps juvenile (up to 15 cm long) and larvae were collected and flash frozen in Tissue-Tek^® O.C.T TM embedding medium (Sakura Finetek USA Inc., Torrance, CA, USA). Sagittal cryosections (20 μm) of the eyes were obtained using CM1850 UV cryostat (Leica Biosystems Nussloch GmbH, Nussloch, BW, Germany) and collected on ColorFrost Plus microscope slides (Thermo Fisher Scientific, Pittsburgh, PA, USA), fixed in 3% paraformaldehyde (Sigma-Aldrich Inc., St. Louis, MO, USA) and stored at −80°C for further use.

Eight juvenile fish were kept in a 75 Gallon glass aquarium with UV filter and standard fluorescent light (40 watts); fish were kept in dechlorinated tap water at 28°C with aeration and biological and mechanical filtration and fed daily with fish ration. Fish were exposed to a day and night cycle of 12 h for 30 days followed by euthanasia, according to Fuller and Claricoates (Fuller and Claricoates, 2011 (link); Supplementary Video 2).
Sagittal sections (20 μm) were obtained using CM1850 UV cryostat (Leica Biosystems Nussloch GmbH, Nussloch, BW, Germany), placed on ColorFrost Plus microscope slides (Thermo Fisher Scientific, Pittsburgh, PA, USA) and stored at −80°C for further use. Eight wild-caught juvenile fish were used as control for these experiments.