Figure 5

Bipolar birthdating in the postnatal mouse retina using combined tritiated-thymidine and immunofluorescence to determine bipolar type. Immunofluorescent staining of dissociated P16 retinas with (a) anti-glutamine synthetase (Muller glia), (c) Rho4D2 (rods), (e) anti-Chx10 and anti-PKC-positive (yellow; rod bipolar cells), and (g) anti-Chx10 and and anti-PKC-negative (red; cone bipolar cells). Blue nuclear stain is DAPI. The arrow in (e) shows a triple stained rod bipolar with blue (DAPI) nuclei, also stained with Chx10 (red), leaving a purple nuclei, and also with PKC+ (green) processes. The arrow in (g) shows a cone bipolar cell with a purple nucleus stained only for DAPI (blue) and Chx10 (red), and PKC-negative processes. (b, d, f, h) Autoradiography of [3H]thymidine from the same fields show birthdated cells, which have a high density of silver grains (arrows and arrowheads). Birthdated Muller glia are indicated by the arrow in (a, b), non-Muller glia by the arrowhead in (a, b), rod by the arrow in (c, d), non-rod by the arrowhead in (c, d), rod bipolar cell by the arrow in (e, f), and cone bipolar cell by the arrow in (g, h). (i) The percentage of heavily labeled cells of the indicated cell type (that is, interpreted as cells born) on P0, P2, P4 and P6 was scored on each retina. (j) The percentage of rod versus cone bipolar type was further divided. P0, n = 3 retinas; P2, n = 8 retinas; P4, n = 6 retinas; and P6, n = 5 retinas.