Fig. 5. Wild-type (A and B) and Crygbnop (C and D) embryos at E17.5 were stained with antibodies to fibrillarin (green channel) and counterstained with propidium iodide (red channel). In the wild-type lens (A), fibrillarin adopts an open floret staining pattern in those nuclei that are transcriptionally active (B, arrow). This pattern then changes in the differentiated fibre cells, resulting in a compact spot(s), indicating that transcriptional activity is reduced (B, asterisk). In the Crygbnop E17.5 lens (C), the fibrillarin pattern is punctate when compared with the wild-type lens (A). Many of the nuclei appear to have shut down transcription, as shown by the intense coalesced staining for fibrillarin (C and D, asterisk). Some Crygbnop nuclei retain an open staining of fibrillarin (D, arrow), whilst in the nuclei containing prominent γBnop-crystallin inclusions (arrowheads), the fibrillarin is no longer present in foci. This staining pattern was not observed in the wild-type lens. Scale bars = 50 µm in (A and B) and 10 µm in (C and D). (E) Immunoblotting with antibodies to vimentin, CP49, filensin, actin, αB-crystallin, αA-crystallin, β-crystallin and γ-crystallin were used to compare levels in E18.5 eye samples from wild-type (+/+), heterozygote (N/+) and homozygote (N/N) embryos. Protein levels appear largely unaffected in all cases except for CP49 and filensin. CP49 and filensin were detected in the N/+ and N/N samples with higher protein loading levels, indicating that they are still expressed although at significantly reduced levels. Cryosections from E17.5 Crybnop mice were stained with antibodies to HSP70 and αB-crystallin (F and G, respectively; green channel) and counterstained with propidium iodide (red channel). Some inclusions are stained throughout with the antibodies to the protein chaperones (arrows), whereas in some instances only the periphery of the deposits is stained (arrowheads). Scale bars = 5 µm.