`Semifree-floating' treatment: a simple and fast method to process consecutive sections for immunohistochemistry and neuronal tracing

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Abstract

A method is described which allows for histochemical processing of thick (50–200 μm) and consecutive sections of neural tissue, a prerequisite for many neuroanatomical studies. Two examples are given: (A) biotin-dextran-amine (BDA) tracing of neuronal connections in 50–100 μm thick vibratome sections of the adult rat brain and (B) immunohistochemical analysis of tyrosine hydroxylase-positive bulbospinal fibers in 50 μm thick cryosections of spinal cord.

Introduction

Neuroanatomical studies often rely on an accurate three-dimensional reconstruction of the investigated neurons or tissue. Tracing of neuronal pathways and reconstruction of the spatial relationships of different cell populations allow the investigator to understand the circuitry of the central nervous system and deduce functional properties. Today, histochemical tools allow not only visualisation of cells and cellular structures but with the advent of immunohistochemistry and modern neuronal tracers it has become possible to demonstrate many subcellular structures (synaptic vesicles, synaptic boutons, cytoskeleton, myelin, etc.) and molecular constituents of neuronal tissues (i.e. neurotransmitters, defined proteins of the cell body, cell surface or extracellular matrix) by light microscopic means.

The three-dimensional reconstruction of tissues is usually achieved by sectioning the area of interest, mounting these sections in a consecutive order on slides, processing them with the required markers, antibodies and/or histochemical reactions, and then viewing the sections in order to get an impression of the spatial relations of the investigated tissue sample. Modern computer-based imaging techniques allow an easy creation of 3D-rendered digital images (Kaufman, 1990). The number of sections required is determined by the dimensions of the area of interest (which in tracing studies can be several centimetres) and the thickness of the sections. It is obvious that in order to limit the number of sections it is desirable to increase the section thickness to a maximum. However, when high molecular weight markers (such as antibodies or avidin-horseradish peroxidase) are used to detect molecules of interest, penetration of these markers into the section becomes problematic. Even when the penetration into the tissue is satisfying, subsequent washing out of excess (unbound) marker is often insufficient, resulting in undesirable high background staining.

To avoid these problems, sections thicker than 30 μm are often processed as `free-floating' sections. This means they are not taken onto slides immediately upon sectioning but collected in a buffer and processed freely-floating. Thus, the reaction solutions have access to the tissue from both surfaces of the section and can diffuse more easily into the tissue. Also, the washing-out of excess, unbound marker is facilitated. The disadvantages of this technique are the large amounts of (often expensive) reagents needed and the loss of the consecutive order of the sections if they are processed together in the same vessel to reduce the amount of reaction solutions. Also, the transfer of the processed free-floating sections onto slides is very tedious and time-consuming, and the sections easily are damaged.

In this paper we present a fast and simple technique that ensures good penetration of markers and washing solutions into the tissue sections, while maintaining their consecutive order and avoiding later manipulation of the processed sections. It thus combines the advantages of both the slide-bound and the free-floating processing of thick tissue sections. For obvious reasons we call it the `semifree-floating' technique.

Section snippets

Animals, surgery, and tissue preparation

In all experiments young adult Lewis rats from our own breeding colony were used. Rats were anaesthetised by a combination of Hypnorm (0.3 mg/kg) and midazolam (0.6 mg/kg) given intraperitoneally. A laminectomy was performed at level T8 and the spinal cord transsected partially as described in Schnell and Schwab (1993). The scalp was opened and a hole drilled into the skull. Using a Hamilton syringe 2.5 μl of a 10% BDA (10 000 MW; Molecular Probes, Eugene, OR) solution in 0.01M phosphate buffer

Results and discussion

The `semi-freefloating' processing of sections yielded staining of labelled structures comparable to freefloating processing with the advantage of keeping the sections in the right sequence. Undesirable high background did not occur (Fig. 2)

Acknowledgements

We thank Lisa Schnell, Drs. Josef Kapfhammer, Wendy Tillotson, and Martin Schwab for critically reading the manuscript and many helpful suggestions. The laboratory of Dr. M.E. Schwab, where the presented work was carried out, is supported by the Swiss National Science Foundation (grant no. 31-29981.90), the American Paralysis Association (Springfield, New Jersey), The International Research Institute for Paraplegia (Zurich), the Paul Schiller Foundation (Zurich), and an anonymous donation

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