Calretinin and calbindin-28 kDa immunofluorescence is not present in the distal dendritic compartment of retinal ganglion cells as seen in retinal whole-mounts

Double labeling for calretinin (red) and microtubule-associated protein 1 (MAP-1; green) in the ganglion cell layer (GCL)/nerve fiber layer (NFL) shows that MAP-1 positive dendrites are not colabeled with calretinin. Some large retinal ganglion cells (RGCs) that are completely ringed by MAP-1 staining (arrowhead) are not positive for calretinin. RGCs with smaller somata partially ringed with MAP-1 staining are immunopositive for calretinin (arrows). Other brightly stained somata not showing MAP-1 immunofluorescence are the displaced amacrine cells that also stained for calretinin. Confocal plane showing that the MAP-1 positive (green) dendrites (arrow) do not merge with the calretinin-positive plexus (red) in the inner plexiform layer. Double labeling for calbindin-28 kDa (red) and MAP-1 (green) in the GCL/NFL shows that some large RGCs that are completely ringed by MAP1 staining (arrowhead) are positive for calbindin-28 kDa. Some RGCs with smaller somata that are incompletely ringed with MAP-1 are also calbindin-28 kDa positive while others are not (arrow). Some neurons incompletely ringed with MAP-1 are also not calbindin-28 kDa positive (arrow). Scale bar represents 20 µm.<p><b>Copyright information:</b></p><p>Taken from "Subcellular compartmentalization of two calcium binding proteins, calretinin and calbindin-28 kDa, in ganglion and amacrine cells of the rat retina"</p><p></p><p> 2008;14():1600-1613.</p><p>Published online 31 Aug 2008</p><p>PMCID:PMC2528027.</p><p></p

Calretinin and calbindin-28 kDa are differentially distributed in amacrine cells as seen in retinal whole-mounts

Double labeling for calretinin (red) and calbindin-28 kDa (green) in the inner nuclear layer shows that labeling for each was present in a distinct set of amacrine cells For the apparent region of overlap (arrowhead) in the overlay of this confocal plane, examination of different z-planes revealed that these were disparate cells located at different depths. Arrows indicate calbindin-28 kDa-immunopositive cells. Scale bar represents 20 µm. Abbreviations: bv is blood vessel.<p><b>Copyright information:</b></p><p>Taken from "Subcellular compartmentalization of two calcium binding proteins, calretinin and calbindin-28 kDa, in ganglion and amacrine cells of the rat retina"</p><p></p><p> 2008;14():1600-1613.</p><p>Published online 31 Aug 2008</p><p>PMCID:PMC2528027.</p><p></p

Comparison of immunofluorescence for the calcium binding proteins, calretinin and calbindin-28 kDa, and voltage-gated sodium channel antibodies in the ganglion cell layer (GCL)/nerve fiber layer (NFL) as seen in retinal whole-mounts

Calcium binding proteins (CBP) in the NFL are extensively colocalized with Pan-NaV. Some Pan-Na stained retinal ganglion cell (RGC) somata were also immunopositive for CBPs (arrowhead) while others were not (double arrows). Initial segments of RGCs, (arrow) some of which can be seen emerging from the RGC somata, were immunopositive for Pan-Na but not colabeled with CBPs. Na1.1-immunopositive (green) RGC nerve fiber bundles in the nerve fiber layer (NFL) were colabeled with CBPs (red), but the axon initial segments (arrow) were not. Na1.2 immunopositive (green) RGC nerve fiber bundles in the NFL were colabeled with CBPs (red) but not the axon initial segments (arrow). Na1.6 immunopositive (green) axon initial segments (arrow) were not colabeled with CBPs (red). Scale bar equals 20 µm. Abbreviations: bv is blood vessel.<p><b>Copyright information:</b></p><p>Taken from "Subcellular compartmentalization of two calcium binding proteins, calretinin and calbindin-28 kDa, in ganglion and amacrine cells of the rat retina"</p><p></p><p> 2008;14():1600-1613.</p><p>Published online 31 Aug 2008</p><p>PMCID:PMC2528027.</p><p></p

Calretinin and calbindin-28 kDa are distinctly distributed in the ganglion cell and nerve fiber layer as seen in retinal whole-mounts

Double labeling for calretinin (red) and calbindin-28 kDa (green) in the ganglion cell layer (GCL)/nerve fiber layer (NFL) shows that labeling for each was present in a distinct set of neurons. Calbindin-28 kDa positive cell bodies are indicated by arrowheads. Some calretinin-positive neurons show processes (arrow) that ascend distally. Note the discontinuous staining pattern of calretinin in the NFL in contrast to a smoother staining pattern for calbindin-28 kDa. , A single confocal optical section distal to that of shows that the calretinin positive processes (arrow) are directed in the inner plexiform layer (IPL) distally toward a calretinin-immunopositive plexus, a characteristic of displaced amacrine cells. Representative calretinin and calbindin-28 kDa double staining in the GCL/NFL is shown. Channel intensity profiles for the red and green channels for straight lines along the long axis (lines a and b in shown in and respectively) show different intensity profiles for calretinin and calbindin-28kDa immunofluorescence. Scale bar represents 20 µm.<p><b>Copyright information:</b></p><p>Taken from "Subcellular compartmentalization of two calcium binding proteins, calretinin and calbindin-28 kDa, in ganglion and amacrine cells of the rat retina"</p><p></p><p> 2008;14():1600-1613.</p><p>Published online 31 Aug 2008</p><p>PMCID:PMC2528027.</p><p></p

Calretinin, calbindin-28 kDa, and NF-200 kDa immunofluorescence in the ganglion cell layer (GCL)/nerve fiber layer (NFL) as seen in retinal whole-mounts

Double labeling for calretinin (red) and NF-200 kDa (green) shows retinal ganglion cell (RGC) somata, surrounded peripherally by light NF-200 kDa immunofluorescence (arrows), that were not immunopositive for calretinin. Calretinin immunofluorescence was present at discrete locations intermittently along the long axis of the RGC nerve fiber, whereas the NF-200 kDa immunofluorescence was uniform. Channel intensity profiles for the red and green channels along the long axis (lines a and b in shown in and respectively) revealed that for regions on the long axis where staining for calretinin was prominent, staining for NF-200 kDa was less prominent () and vice versa (). Double labeling for calbindin-28 kDa (red) and NF-200 kDa (green) showed that calbindin-28 kDa-positive immunofluorescence was smoothly distributed in the nerve fibers similar to NF-200 kDa. RGC somata that were surrounded peripherally by light NF-200 kDa immunofluorescence (arrow) were also stained with calbindin-28 kDa while for others (arrowhead) staining was less prominent. Channel intensity profiles for the red and green channels for straight lines along the long axis (lines d and e in shown in and J respectively) presented some region where immunofluorescence for NF-200 kDa was prominent while that for calbindin-28 kDa was less prominent () and others where the intensity profiles were similar (). Scale bar represents 20 µm.Abbreviations: bv is blood vessel.<p><b>Copyright information:</b></p><p>Taken from "Subcellular compartmentalization of two calcium binding proteins, calretinin and calbindin-28 kDa, in ganglion and amacrine cells of the rat retina"</p><p></p><p> 2008;14():1600-1613.</p><p>Published online 31 Aug 2008</p><p>PMCID:PMC2528027.</p><p></p

Calretinin and calbindin-28 kDa immunolabeling of a vertical cryosection

Calretinin immunolabeling was present in cell bodies and processes of amacrine cells at the inner nuclear layer (INL)-inner plexiform layer (IPL) border. Calretinin labeling was also present in cell bodies and processes (arrows) in the ganglion cell layer (GCL). Calretinin labeling is also found in three distinct bands in the IPL and retinal ganglion cell (RGC) axons in the nerve fiber layer (NFL; arrowhead). Calbindin-28 kDa immunolabeling was present in cell bodies and processes of horizontal cells at the outer plexiform layer (OPL)-INL border. Calbindin-28 kDa also labeled amacrine cells at the INL-IPL border. Some descending processes were seen for some of these neurons (arrow). There are also diffuse calbindin-28 kDa-positive punctate in the IPL. Calbindin-28 kDa labeling is seen in few cells in the GCL as well as in the RGC axons in the NFL (arrowhead). Scale bars represent 20 µm.<p><b>Copyright information:</b></p><p>Taken from "Subcellular compartmentalization of two calcium binding proteins, calretinin and calbindin-28 kDa, in ganglion and amacrine cells of the rat retina"</p><p></p><p> 2008;14():1600-1613.</p><p>Published online 31 Aug 2008</p><p>PMCID:PMC2528027.</p><p></p

Size distributions of Calcium-binding protein (CBP) immunopositive and immunonegative neurons are shown

The histogram shows the surface area of projection of the somata of neurons in the retinal ganglion cell (RGC) layer from retinal whole mounts that were immunopositive for Pan-Na only (n=40, red) or Pan-Na and calcium binding proteins (CBP; n=40, black). The histogram is based on projections of all optical planes corresponding to the RGC layer from five midperipheral retinal areas (256 μm × 256 μm).<p><b>Copyright information:</b></p><p>Taken from "Subcellular compartmentalization of two calcium binding proteins, calretinin and calbindin-28 kDa, in ganglion and amacrine cells of the rat retina"</p><p></p><p> 2008;14():1600-1613.</p><p>Published online 31 Aug 2008</p><p>PMCID:PMC2528027.</p><p></p

Measurements of retinal degeneration.

<p>(A) Counts of nuclei per column of nuclei in the outer nuclear layers (ONL) of retinas from mice with different numbers of wild type rhodopsin genes. Genotypes are abbreviated on the curves: 0 Rho is mRho^−/− (filled squares), 1 Rho is mRho^+/− (open circles), 2 Rho is mRho^+/+ (filled circles), 3 Rho is mRho^+/+NHRE^+/0 (open triangles), and 4 Rho is mRho^+/+NHRE^+/+ (open squares). (B) Counts of nuclei per column of nuclei in the ONL of retinas from mice with the P23H-rhodpsin transgene. Genotypes are abbreviated on the curves: 1 Rho+P23H is mRho^+/−P23H^+/0 (open circles), 2 Rho+P23H is mRho^+/+P23H^+/0 (filled circles), and 3 Rho+P23H is mRho^+/+NHRE^+/0P23H (open triangles). We counted 60–100 columns of nuclei for multiple areas within each retina (3 eyes from 3 individual mice per genotype per timepoint) and averaged them for each time point. Error bars indicate standard error of the mean. Curves were fit to an exponential decay curve, allowing for a plateau value. Exponentials are from non-linear curve fitting using the Marquardt-Levenberg method in Origin, with weighting by 1/variance. The 3 Rho curve-fitting also included data from 3 mice at 8.5 months, which had 6.7 nuclei, and the 4 Rho curve-fitting included data from 1 mouse at 10 months, which had 1.5 nuclei (not shown).</p

Rhodopsin expression from mRho and NHRE alleles.

a<p>For the spectrophotometric analysis of rhodopsin protein concentration, we performed a univariate ANOVA with a Least Squares Difference post-hoc analysis. For 1 versus 2 and 3 copies, <i>P</i> = 0.002 and 0.006, respectively. Two copies of mouse and two copies of human (NHRE) are not significantly different.</p

Hematoxylin and eosin stained images of retinal sections from mice with different numbers of wild type rhodopsin genes.

<p>Genotypes are indicated at left, where 1 Rho is mRho^+/−, 2 Rho is mRho^+/+, and 3 Rho is mRho^+/+NHRE^+/0. Ages in months are indicated at the top. Retinal layers are abbreviated at right, where ROS is rod outer segment, RIS is rod inner segment, ONL is outer nuclear layer, OPL is outer plexiform layer, INL is inner nuclear layer, and IPL is inner plexiform layer.</p