title: 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.
authors.affiliation: Department of Ophthalmology, the Second Hospital of Jilin University, Changchun, China.
authors.forename: Jinling
authors.initials: J
authors.lastname: Fu
authors.forename: Wei
authors.initials: W
authors.lastname: Fang
authors.forename: Jian
authors.initials: J
authors.lastname: Zou
authors.forename: Ming
authors.initials: M
authors.lastname: Sun
authors.forename: Kira
authors.initials: K
authors.lastname: Lathrop
authors.forename: Guanfang
authors.initials: G
authors.lastname: Su
authors.forename: Xiangyun
authors.initials: X
authors.lastname: Wei
mesh_terms: D000818:Animals; D001399:Azure Stains / Q000032:analysis; D002467:Cell Nucleus / Q000648:ultrastructure*; D004396:Coloring Agents / Q000032:analysis*; D008751:Methylene Blue / Q000032:analysis; D008853:Microscopy / Q000379:methods; D009499:Neutral Red / Q000032:analysis; D020419:Photoreceptor Cells, Vertebrate / Q000648:ultrastructure*; D016613:Plastic Embedding / Q000379:methods; D012160:Retina / Q000166:cytology* / Q000648:ultrastructure; D012394:Rosaniline Dyes / Q000032:analysis; D013194:Staining and Labeling / Q000379:methods*; D015027:Zebrafish / Q000033:anatomy & histology*
chemical_list: D001399:Azure Stains; D004396:Coloring Agents; C007722:Feulgen stain; D012394:Rosaniline Dyes; D009499:Neutral Red; D008751:Methylene Blue