A robust procedure for distinctively visualizing zebrafish retinal cell nuclei under bright field light microscopy

Abstract

To simultaneously visualize individual cell nuclei and tissue morphologies of the zebrafish retina under bright field light microscopy, it is necessary to establish a procedure that specifically and sensitively stains the cell nuclei in thin tissue sections. This necessity arises from the high nuclear density of the retina and the highly decondensed chromatin of the cone photoreceptors, which significantly reduces their nuclear signals and makes nuclei difficult to distinguish from possible high cytoplasmic background staining. Here we optimized a procedure that integrates JB4 plastic embedding and Feulgen reaction for visualizing zebrafish retinal cell nuclei under bright field light microscopy. This method produced highly specific nuclear staining with minimal cytoplasmic background, allowing us to distinguish individual retinal nuclei despite their tight packaging. The nuclear staining is also sensitive enough to distinguish the euchromatin from heterochromatin in the zebrafish cone nuclei. In addition, this method could be combined with in situ hybridization to simultaneously visualize the cell nuclei and mRNA expression patterns. With its superb specificity and sensitivity, this method may be extended to quantify cell density and analyze global chromatin organization throughout the retina or other tissues.

Figure 1.

Schematic drawings illustrating how the thickness of tissue sections and cytoplasmic background signals may affect the imaging effects of two closely juxtaposed cell nuclei under bright field light microscopy. (A) In thick sections, the images of two closely juxtaposed cell nuclei may merge together from the top view. (B) In thin sections, the images of two closely juxtaposed cell nuclei will appear separated from each other from the top view. (C) Weak nuclear signals and high cytoplasmic signals may make the images of the two nuclei appear merged from the top view.

Figure 2.

Effects of section thickness on nuclear distinguishability. (A–D) Individual nuclei show more clear boundaries in 2-μm sections (A) than in 3-μm (B), 4-μm (C), and 6-μm (D) sections. Arrows indicate the image overlapping of neighboring nuclei.

Figure 3.

Effects of HCl hydrolysis conditions and Schiff reaction durations on Feulgen staining. (A–J) More concentrated HCl or longer incubation gave stronger Feulgen staining than less concentrated HCl or shorter incubation. (K–M) Feulgen staining increased with prolonged Schiff reaction, reaching a maximal intensity after 2 hrs incubation (data not shown). (N) The chemical reactions of HCl hydrolysis of the DNA molecules and Schiff reaction (Pearse 1968, 1972; Kiernan 2008). RPE, retinal pigment epithelium; ONL, outer nuclear layer; INL, inner nuclear layer; RGC, retinal ganglion cell layer.

Figure 4.

Feulgen staining is sensitive enough to distinguish the euchromatic and heterochromatic regions in the cone nuclei in the zebrafish retina. (A) The euchromatic regions in cones, but not rods, are clearly distinguishable from heterochromatic regions in 2-μm JB-4 sections after Feulgen staining. (B) A transmission electron micrograph of the outer nuclear layer of the adult retina shows the heterochromatin and euchromatin in both rods and cones. UV, UV cone nuclei; long cone, either a green, red, or blue cone nucleus.

Figure 5.

Feulgen staining provides a more specific and stable staining than methylene blue-azure II and nuclear fast red staining. (A–B) Methylene blue-azure II and nuclear fast red staining showed strong cytoplasmic background. Destaining washes nonspecifically removed both nuclear and cytoplasmic signals. After an overnight (o/n) wash, methylene blue-azure II staining was completely removed. (C) Covalent Feulgen nuclear staining remained largely unaffected after an overnight wash. RPE, retinal pigment epithelium; ONL, outer nuclear layer; INL, inner nuclear layer; RGC, retinal ganglion cell layer.

Figure 6.

Feulgen staining is compatible with alkaline phosphatase-based colorimetric detection that is frequently used in conventional whole-mount in situ hybridization. (A–B) Both the blue in situ hybridization signals (arrows) and Feulgen staining were visible. A peripheral retinal region is magnified 3 times in B. (C–D) Feulgen staining of regular 5-dpf fish that was not pretreated with the in situ hybridization showed more distinguishable nuclear boundaries. A peripheral retinal region is magnified 3 times in D.