Polyfreeze tissue freezing media red

Lenses are isolated and fixed for 2 or 24 hrs at 4°C in 4% paraformaldehyde, washed in PBS, and then cryoprotected (30% sucrose) prior to freezing in Polyfreeze Tissue Freezing Media Red (Polyscience #25115). 20μm thick serial cryosections were cut using a Microm HM 550 Cryostat, and only the central sections used for these studies. Immunolabeling was performed as described previously [41 (link)]. Briefly, the sections were permeabilized with 0.25% Triton X-100 in PBS buffer (Corning) for 30 min and the incubated in block buffer (5% goat serum, 1% BSA in PBS) for 1 hr prior to incubation in primary antibody for either 3 hrs at 37 C or overnight at 4 C, followed by incubation with secondary antibody for 2 hrs at 37 C (Jackson ImmunoResearch Laboratories). F-actin was localized with Alexa 647-conjugated phalloidin (Invitrogen-Molecular Probes). Nuclei were stained with DAPI (Biolegend). For histological analyses, lenses were fixed in 4% paraformaldehyde, cryoprotected (30% sucrose) for 24 hrs and frozen with tissue freezing media prior to cryosectioning. Sections were stained with hematoxylin and eosin.
Images were acquired with a Nikon Eclipse Ti microscope with a Nikon Photometrics Cool Snap HQ camera.