Technovit 7100 glycol methacrylate

Eyes were harvested at appropriate time points and fixed in half strength Karnovsky fixative (4% paraformaldehyde + 2.5% glutaraldehyde) in 0.1M PO₄ buffer pH 7.4 for 48 hours, dehydrated in graded ethanol and embedded in Technovit 7100 Glycol Methacrylate (Electron Microscopy Sciences, Hatfield, PA). Embedded tissues were serially sectioned (2 μm) by passing through the optic nerve head and stained with Hematoxylin and Eosin (H&E). For each retina a section cut along the naso-temporal equator containing the optic nerve head was acquired by an upright Axiophot microscope (Zeiss, Germany) equipped with a Plan Neofluar 20X objective (NA 0.5). A blinded investigator quantified the thickness of the outer and inner nuclear layers and of the inner plexiform layer at ten locations equally spaced along the naso-temporal axis, and values for a given retina were expressed either as average number of nuclei (for nuclear layers) or average microns (for the inner plexiform layer).

Microtissues within agarose hydrogels were fixed in 10% neutral buffered formalin (No. 225; Fisher Scientific, Agawam, MA) for a minimum of 24 h prior to dehydration and processing. Microtissues within the agarose molds were then embedded in Technovit 7100 glycol methacrylate (No. 14653l; Electron Microscopy Sciences, Hatfield, PA) per manufacturer’s specifications with one modification. Samples were partially submerged in the infiltration solution and hardener 2 mixture to allow the sample to firmly attach to the bottom of the histology mold prior to filling the mold completely and adding a block holder. Samples were sectioned at a thickness of 3 μm, after which sections were mounted on slides and stained with hematoxylin and eosin (H&E) for histological examination.