Mice were anesthetized and perfused with phosphate buffered saline pH 7.4 (PBS) prior to enucleation. Eyes from all three species were hemisected in PBS and immediately fixed in PFA in the following conditions: ground squirrel and macaque eyes were fixed in 4% PFA for fifty minutes on ice. Mouse eyes were fixed in 4% PFA for fifty minutes on ice (DSCAM staining) or 4% PFA for 30 minutes at room temperature (all other staining). Tissue was sunk in 30% sucrose overnight prior to freezing. Tissue was then immersed in tissue-tek optimal cutting temperature media (Sakura Finetek, USA) and rapidly frozen by holding the tissue above the liquid phase of liquid nitrogen. Sections were cut at 10 μm on to charged slides.
Tissue tek optimal cutting temperature media
Manufactured by Sakura Finetek
Sourced in United States
Tissue-Tek Optimal Cutting Temperature (O.C.T.) Compound is a specially formulated embedding medium designed for cryosectioning of tissue samples. It is used to support and protect the structural integrity of frozen tissue specimens during the sectioning process, enabling thin, uniform sections to be cut on a cryostat.
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2 protocols using tissue tek optimal cutting temperature media
1
Tissue Preparation for Eye Immunofluorescence
2
Liver Tissue Preparation for Imaging
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Adult and 2wk animals were sacrificed and tissues fixed by intracardiac perfusion with cold PBS followed by cold 4% paraformaldehyde (PFA; Thermo Fisher; 41678–5000) using a peristaltic pump (Kamoer DIpump550). Embryos and neonatal mice under 10 days of age were not perfused. Instead, livers were dissected, swirled in Dulbecco’s Phosphate-Buffered Saline (DPBS; Thermo Fisher; 14190250) to wash off excess blood, and drop fixed in cold 4% PFA. For all samples, dissected livers were fixed overnight in 4% paraformaldehyde at 4°C. The next day, tissues for whole mount imaging were moved to 0.05% sodium azide (Ricca Chemical; RC7144.8) in PBS at 4°C until further processing. Tissues for conventional histology were moved to 30% sucrose overnight, then embedded in Tissue-Tek Optimal Cutting Temperature media (Sakura Finetek USA, Torrance, CA) and frozen on dry ice. Conventional histology tissues and sections were stored at –80°C until use.
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