Histochemistry on vibratome sections of fish tissue: a comparison of fixation and embedding methods

B Grunow,T Kirchhoff,S Harzsch,T Moritz,T Lange

doi:10.3354/ab00624

Abstract

Despite improvements in imaging techniques during recent years, for many non- model systems the fixation of tissues followed by embedding and sectioning for histochemical or immunohistochemical staining remains an important technique in vertebrate histology. The pres- ent study sets out to explore the preservation of histological sections of fish tissues using different preparation techniques. The quality of transverse vibratome sections from trunk segments of the lesser-spotted dogfish Scyliorhinus canicula, Atlantic sturgeon Acipenser oxyrinchus and zebrafish Danio rerio were compared using different fixatives (formaldehyde, paraformaldehyde and zinc-formaldehyde) and embedding methods (gelatine, agarose and low-temperature melting agarose). Our data show that the quality of the vibratome sections for histochemical staining is strongly dependent upon fixation and embedding media. Although paraformaldehyde fixation results in a more pronounced shrinkage of the trunk segment than the other fixatives used , the quality of the sections and the histochemical staining was best with this fixative in zebrafish and dogfish. Additionally, the embedding methods have a strong influence on the quality of the sec- tions. In the dogfish and sturgeon samples, the preferred embedding media were agarose and low-temperature melting agarose, since gelatine often caused shrinkage of the tissues. In conclu- sion, for histochemical examinations, the processing protocols for vibratome sectioning need to be adapted individually to each study organism.

Highlights

Methods for studying mammalian tissue using vibratome sections have been commonly used since the early 1970s (Hökfelt & Ljungdahl 1972, Lindvall et al 1973), and from the 1980s, publications on vibratome sections using fish tissue are available (Funch et al 1984, Kah et al 1986, Yulis & Lederis 1986)
Our data show that the quality of the vibratome sections for histochemical staining is strongly dependent upon fixation and embedding media
The goal of this study was to explore the effects of different fixation and embedding protocols on the quality of vibratome sections and fluorescence histochemistry in 3 different fish species

Summary

Introduction

Methods for studying mammalian tissue using vibratome sections have been commonly used since the early 1970s (Hökfelt & Ljungdahl 1972, Lindvall et al 1973), and from the 1980s, publications on vibratome sections using fish tissue are available (Funch et al 1984, Kah et al 1986, Yulis & Lederis 1986) This technique is well-suited to prepare tissue sections, typically between 10 and 100 μm thick, that can subsequently be processed by histochemical, immunohistochemical or electron microscopic methods. The FOR-fixed and AGAembedded sturgeon samples showed a massive degradation in their morphology (Fig. 7I−L). In these sections, structural details were only poorly resolved (Table 4)

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