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. 2011 Jan;6(1):46-55.
doi: 10.1038/nprot.2010.165. Epub 2010 Dec 16.

Embedding, serial sectioning and staining of zebrafish embryos using JB-4 resin

Jessica Sullivan-Brown  1 , Margaret E Bisher, Rebecca D Burdine
Affiliations

Embedding, serial sectioning and staining of zebrafish embryos using JB-4 resin

Jessica Sullivan-Brown et al. Nat Protoc. 2011 Jan.

Abstract

Histological techniques are critical for observing tissue and cellular morphology. In this paper, we outline our protocol for embedding, serial sectioning, staining and visualizing zebrafish embryos embedded in JB-4 plastic resin-a glycol methacrylate-based medium that results in excellent preservation of tissue morphology. In addition, we describe our procedures for staining plastic sections with toluidine blue or hematoxylin and eosin, and show how to couple these stains with whole-mount RNA in situ hybridization. We also describe how to maintain and visualize immunofluorescence and EGFP signals in JB-4 resin. The protocol we outline-from embryo preparation, embedding, sectioning and staining to visualization-can be accomplished in 3 d. Overall, we reinforce that plastic embedding can provide higher resolution of cellular details and is a valuable tool for cellular and morphological studies in zebrafish.

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Figures

Figure 1
Figure 1
Timeline of histological protocol using JB-4™ resin.
Figure 2
Figure 2
Visual aid for embedding and sectioning zebrafish embryos in JB-4™ resin. (A) This is the set-up during the polymerization reaction of JB-4™ resin. A single polyethylene mold with a white plastic embedding stub in a metal casting tray covered with plastic wrap. The inset shows the mold and embedding stub before the specimen and JB-4™ resin are added. (B) During the second embedding step, the solid resin block is removed from the chuck with a coping saw. The chuck is first secured by a table vice before the resin block is removed. (C) Before sectioning, the resin block is trimmed with a razor blade so that the cutting surface will be in a trapezoid shape (trapezoid surface is traced with a gray line in the inset). (D) The set-up for sectioning on the Leica microtome. The embedding stub is clamped on the microtome. Below the embedding stub is the glass knife that is used for sectioning. (E) Before the sections are collected, rows of water droplets are placed on a glass slide with a syringe and needle. (F) Collect each section individually. Each section will stay on the glass knife after it is cut. The section is removed from the glass knife with forceps by gently picking it up at a corner and placing it on the water droplet.
Figure 3
Figure 3
Toluidine blue and Hematoxylin and Eosin staining on JB-4™ plastic sections. (A–C) Toulidine blue staining on transverse sections through the head (A), ear (B) and gut (C) regions of a 5 day wild-type embryo; Toulidine blue stains the nucleus a dark blue, the cytoplasm a light blue and the cartilage a dark pink color. (D–F) H&E staining on consecutive transverse sections through the head (A), ear (B) and gut (C) regions of the same 5 day wild-type embryo shown in A–C; H&E stains the nucleus a dark purple and the cytoplasm pink. (G–H) Consecutive longitudinal sections of a 4 day wild-type embryo stained with Toluidine blue (G) and H&E (H). (I–J) Sections through the notochord to show the typical amount of shrinkage/swelling seen with this procedure. The diameter indicated with the black line in each image was measured using ImageJ software. (I) Anterior notochord of a 5 day wild-type embryo. Notochord diameter is 56 microns. (J) Posterior notochord of a 2 day wild-type embryo. Notochord diameter is 59 microns. All sections are 4μm. A–B, D–E and G–H were taken were taken at 10× magnification, C,F were taken at 20× magnification. I was taken at 32× magnification. J was taken at 40× magnification.
Figure 4
Figure 4
RNA in situ hybridization, Immunofluorescence and GFP signals in JB-4 plastic resin. All images in this figure were produced using this procedure in the Burdine Laboratory. (A–C) RNA in situ hybridization developed with BCIP/NBT in whole-mount and subsequently embedded and sectioned in JB-4™ plastic resin. (A) cmlc2 expression marks the myocardium of the atrial and ventricular chambers of the zebrafish heart. (B) carboxypeptidase A expression marks the exocrine pancreas. (C) Same section as shown in B, stained with an H&E stain. (D–F) Immunofluorescence and GFP visualization in JB-4™ sections. (D) Immunostaining for the sodium potassium pump (α6F antibody, red) and counterstained with Hoechst dye (blue), highlighting the cells of the zebrafish kidney tubule. At these stages the Na+-K+-ATPase pump is expressed in the pronephric duct and is clearly localized to the basolateral surface. Embryos were fixed in Dent's. (E) RNA injection of GAP43-GFP to outline cell membranes. Mosaic expression of GAP43-GFP is preserved in JB-4™ plastic sections at the 2 somite stage. The GFP signal is clearly seen surrounding the cell membranes in this section. (F) cmlc2-GFP transgenic zebrafish strain. In this section, cmlc2-GFP is strongly expressing within the cells lining the ventricular chamber of a 2 day zebrafish heart.
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