Fmr1 transcript isoforms enriched on neuron-specific ribosomes from adult mouse brain.
The neuron-specific, ribosome-bound transcripts were enriched using the RiboTag methodology as described in the text. (A) Immunohistochemistry on paraformaldehyde fixed coronal brain sections from Eno2-Cre:RiboTag mice demonstrating neural specific expression of Rpl22-HA (HA staining); sections are stained with the neuronal marker NeuN and counterstained with the glial specific marker GFAP. Overlap of NeuN and HA staining and absence of overlapping HA and GFAP staining supports neuron-specific labeling of ribosomes. This particular section corresponds to the dentate gyrus area of the hippocampus. Immunohistochemistry in cortex, striatum and other brain regions was similar to the image shown, suggesting that HA staining is specific to neurons. Scale Bar = 40 microns. (B) The Fmr1 transcript isoforms were isolated from immuno-purified polyribosomes and quantified by qRT-PCR as in Fig. 1. The results are averaged values from five independent, neuron-tagged mice. The average degree of neuronal enrichment of the tagged ribosomes over these five experiments was 3.2±0.7 fold, as estimated using the neuron-specific transcript from the NeuN gene (Fig. S4).