Abstract
Purpose :
The inner segment (IS) region of rod photoreceptor neurons is a hub for the synthesis and trafficking of visual proteins. These protein “cargoes” are delivered to the basal body (BB) at the apical edge of the IS region prior to their incorporation into the rod outer segment cilium. The study of the localization and dynamics of IS trafficking processes in single rods with superresolution microscopy techniques requires optimized preparation methods with minimal tissue fixation for thorough antibody penetration and immunolabeling. During such rigorous immunolabeling procedures, the IS membrane is susceptible to membrane extraction. The goal of this project was to develop a new methodology for intensive immunolabeling of mouse rods that both preserves the morphology of the IS and is compatible with superresolution imaging.
Methods :
We lightly fixed mouse retinas with paraformaldehyde (PFA) before a 3-day primary antibody incubation period in a blocking solution containing saponin, a mild detergent. Primary antibodies used targeted syntaxin-3 and Na,K ATPase (ATP1B2), which are IS membrane markers, and phosducin, a cytoplasmic chaperone protein. After a secondary antibody incubation period, we tested different “post” fixation solutions prior to dehydrating the retinas for resin embedding. We imaged ultra-thin retinal sections with structured illumination microscopy (SIM), stochastic optical reconstruction microscopy (STORM) and conventional transmission electron microscopy (TEM).
Results :
We determined that a post-fix solution of 2% PFA and 0.1% glutaraldehyde (w/v) preserved the IS membrane of most rods in immunolabeled retinas as determined by TEM ultrastructure and the SIM/STORM immunolocalization of syntaxin-3 and ATP1B2. We also used STORM to determine that significantly more cytoplasmic phosducin was preserved in the IS of retinas treated with our optimized post-fix solution compared to just PFA alone.
Conclusions :
Our optimization of mouse retinal immunolabeling conditions enables future superresolution localization studies focused on the IS. Specifically, by preserving the IS membrane and cytoplasm, we can more accurately test dynamics of visual protein cargoes with trafficking organelles in the IS and the BB region in future studies.
This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.