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Allen Rodriguez, Luis Pérez de Sevilla Müller, Nicholas Brecha; The RNA binding protein RBPMS is a specific marker of ganglion cells in the mammalian retina. Invest. Ophthalmol. Vis. Sci. 2014;55(13):2392.
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Transcription factors and RNA-binding proteins that are specific to retinal ganglion cells (RGCs) have been reported in the mammalian retina. We report the development and characterization of a new set of antibodies against RNA-binding protein with multiple splicing (RBPMS) that specifically labels the entire RGC population.
Affinity purified polyclonal guinea pig and rabbit antibodies were generated to the N-terminus of RBPMS. The RBPMS polypeptide sequence used for immunization is identical in mouse, rat, monkey and human, and 95% similar for guinea pig. Mouse and rat retinal extracts were evaluated using western blotting. Mouse, rat, guinea pig, rabbit and monkey retinal sections and whole-mounts were evaluated using single and double labeling immunohistochemistry. RBPMS-immunoreactive cells were measured for somatic size and density. Unilateral optic nerve transection or crush was performed in rats and mice.
The guinea pig and rabbit antibodies detected a single band of ~24 kDa on western blots of cell lysates from HEK293T cells transfected with human RBPMS cDNA. No bands were detected in non-transfected lysates. A prominent single band at ~24 kDa was also detected in mouse and rat retinal extracts. Specific RBPMS immunoreactivity was mainly localized to medium to large diameter somata in the ganglion cell layer and to a few medium and large somata at the border of the inner plexiform layer and inner nuclear layer. The size and density of RBPMS immunoreactive cells in mouse and rat retina were comparable to earlier semi-quantitative estimates of RGCs. All Brn3a, SMI-32 and melanopsin immunoreactive RGCs also express RBPMS immunoreactivity. RBPMS immunoreactivity is not expressed in syntaxin (HPC-1) immunoreactive cells in the INL and GCL, consistent with the specificity of RBPMS as a RGC marker. 86% or 98% of RBPMS immunoreactive cells are lost following optic nerve crush or transection at three weeks post injury in mouse and rat, respectively. These findings are consistent with a very weak immunostained band at ~24 kDa in a rat retinal extract collected 56 days after optic nerve transection.
RBPMS antibodies are robust reagents for the identification and quantification of RGCs in multiple mammalian species, and they will be particularly useful for tracking RGCs in chronic disease or acute injury models.
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