Figure 1.
Photoreceptor outer segments immunolabeled with fluorescent secondary antibodies.
Vertical cross sections (1 µm) of A) wallaby retina (“wal”) and B) dunnart retina (“dun”) treated with primary antibodies Rho1D4 (green fluorescent color tag, Alexa 488) and JH492 (red fluorescent color tag, Alexa 594) to visualize the rod opsin and LM-opsin photopigment, respectively. Immunolabeling was restricted to rod (ROS) and cone (arrowheads) outer segments; inner segments of both rods (RIS) and cones (CIS) remained unlabeled. Every cone contains an oil droplet (arrows).
Figure 2.
Alternating immunohistochemical protocols on sequences of consecutive retina sections.
Four sections from a digital stack of A–D) wallaby (“wal”) and E–H) dunnart (“dun”) semi-thin (0.5 µm) horizontal retina sections immunolabeled with primary antibodies Rho1D4 (rod opsin, green) and A and C, E and G) JH455 (S-opsin, red) or B and D, F and H) JH492 (LM-opsin, red). Arrowheads mark the same selection of individual S-cones (gray) and LM-cones (white) in every section. A–D) All cones in this wallaby retina sample immunolabeled with either cone opsin antibody. E–H) Dunnart S-cones display a faint fluorescent label when treated with JH492. A substantial population (51%) of cones remained unlabeled by both opsin antibodies in this sample.
Table 1.
Distribution of cone types in six samples of tammar wallaby retina.
Table 2.
Distribution of cone types in six samples of fat-tailed dunnart retina.
Figure 3.
Outer segments of unlabeled cones in two immunohistochemical protocols.
Four consecutive horizontal sections of A–D) wallaby (“wal”) and E–H) dunnart (“dun”) retina immunolabeled with primary antibodies Rho1D4 (rod opsin, green) and A and C, F and H) JH492 (LM-opsin, red) or B and D, E and G) JH455 (S-opsin, red). A–D) A wallaby cone remained unlabeled (arrow) by both cone opsin antibodies throughout the digital stack because its outer segment is missing. Neighboring cones are labeled by either antibody; their intact outer segments display in the background staining (arrowheads). E–H) The intact unlabeled outer segments of two cones in the dunnart retina (arrow and arrowhead in G) are visible in the background staining. Only one (arrowhead) labeled with a cone opsin antibody in F and H. The other cone (arrow) remained unlabeled in all sections.
Figure 4.
Labeling cone membranes with Peanut Agglutinin, PNA.
A and B) Vertical cross sections (1 µm) of A) wallaby (“wal”) and B) dunnart (“dun”) retina treated with rod opsin primary antibody A) Rho1D4 or B) Rho4D2 (green fluorescence, Alexa 488) and the cone membrane marker PNA (red fluorescence, Streptavidin 594). Rod opsin labels are restricted to rod outer segments (ROS); rod inner segments (RIS) remain unlabeled. Cones contain oil droplets (arrow). A) PNA marks the outer membrane of some cones, not all (arrowhead) in the wallaby retina. B) All cones in the dunnart retina are PNA-positive but staining of outer (arrowhead) and inner segments (CIS) is patchy. C–F) Horizontal sections of C and D) wallaby and E and F) dunnart retina. PNA-positive cone outer segments (arrowheads) have an open chamber where the photopigment C and E) S-opsin and D and F) LM-opsin is located (asterisks).
Figure 5.
Singular co-localizations of rod opsin in a PNA-positive cone outer segment.
Sequence of three consecutive horizontal sections of A–C) wallaby (“wal”) and D–F) dunnart (“dun”) retina treated with rod opsin antibody Rho1D4 (green) and PNA (red). Sections B and E) show the potential co-localization of the rod opsin label in the outer segment chamber of a cone as marked by PNA (red arrow). This co-localization was not observed in the previous A and D) or following C and F) section of the digital stack (white arrows). Neighboring PNA-positive cones are not rod opsin-labeled (arrowheads).
Table 3.
Occurrence of co-localized rod opsin labels in PNA-positive cone outer segments of tammar wallaby retina samples.
Table 4.
Occurrence of co-localized rod opsin labels in PNA-positive cone outer segments of fat-tailed dunnart retina samples.
Figure 6.
Molecular size of rod opsin in three mammalian species.
The rod opsin gene sequence was isolated from retinal mRNA of A) rat and wallaby (“wal”) and B) dunnart (“dun”) by primers, reverse transcribed, amplified by PCR, and visualized by Agarose gel electrophoresis. A single band at approximately 850 bp molecular size displays in all species. Splice isoforms do not occur in the primed sequence of the rod opsin gene. Tissue from a female (♀) and a male (♂) dunnart was used, but delivered the same band. No template controls (NTC) were run for each species (black lanes).