Basic Neuroscience
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Embedding matrix for simultaneous processing of multiple histological samples

https://doi.org/10.1016/j.jneumeth.2012.06.005Get rights and content

Abstract

A method is described for simultaneous histological processing of multiple fixed tissue samples. The tissue samples are embedded in a gelatin–albumin protein matrix that is rapidly solidified and bound to the tissue surface using a cross-linking reagent. After freezing and sectioning, the individual sections containing multiple samples can be processed for immunocytochemical and histochemical staining. The method is demonstrated for simultaneous processing of multiple rodent brains, and for reconstruction of fragmented human postmortem brain samples.

Highlights

► An improved method for embedding tissue in gelatin–agarose matrix is described. ► A cross-linking reagent rapidly solidifies the matrix and binds it to the tissue. ► The procedure allows simultaneous processing of multiple samples. ► A contrast reagent facilitates 3-dimensional reconstructions from digital images. ► The method is shown for multiple rodent brains, and fragmented human brain tissue.

Introduction

Microscopic examination of fixed tissue typically requires labor intensive processing of individual samples. Methods that facilitate simultaneous, high-throughput histological processing reduce the labor costs, and at the same time standardize the staining quality across samples. One commonly used method is to create “tissue microarrays” that embed multiple samples in a single paraffin block for simultaneous sectioning (Kononen et al., 1998). Each section contains multiple samples, and can be stained and analyzed on a single microscope slide. A disadvantage is that the tissue is infiltrated with embedding medium (e.g., paraffin), that require immersion in organic solvents, and this is sometimes incompatible with routine histological procedures, such as immunocytochemical staining of sensitive antigens.

An alternative method for embedding tissue is to use gelatin–albumin to infiltrate or encase tissue samples, with subsequent hardening by exposure to formalin (Snodgress and Dorsey, 1963, Crane and Goldman, 1979). A disadvantage of this method is that it requires lengthy exposure to formalin to solidify the embedding material, up to a week for larger tissue samples.

We describe a modification of the gelatin–albumin method that uses a lysine–glutaraldehyde cross-linking reagent to rapidly solidify the embedding material and bind it to the surface of fixed tissue. The method provides a homogeneous embedding matrix that allows simultaneous processing of multiple tissue samples, and is compatible with a variety of histological methods commonly used on frozen sections of fixed tissue. The matrix also provides support for the tissue, reducing cutting artifacts that commonly occur at the tissue edges while cutting frozen tissue. Additionally, optional inclusion of a color reagent creates contrast with the tissue, and simplifies 3-dimensional computer reconstructions from digital images of the block face taken during sectioning, which is especially useful for complex and fragmented samples.

Section snippets

Materials and methods

Tissue samples were first fixed by exposure to formaldehyde. In the examples shown (Fig. 1, Fig. 2), rodent brains were transcardially perfused with 4% paraformaldehyde in 0.1 M phosphate buffer, pH 7.2, and human brains were cut into coronal slabs about 3 cm thick, and immersed in phosphate buffered 10% formalin for 5 days. All tissue samples were subsequently cryoprotected by immersion in 20% glycerol plus 0.1 M phosphate, pH 7.2 at 4 °C, until they sank to the bottom of the container.

Results

Fig. 1 shows application of this preparation to multiple rodent brains. We have simultaneously processed as many as 20 mouse brains, using 50 × 75 mm microscope slides to accommodate the large sections. Fig. 2 shows an application of the method to human postmortem cerebral cortex. The region containing auditory cortex had been fragmented by the pathologist at autopsy. The pieces were pinned together in their original orientation, and then embedded in matrix and sectioned in a parasagittal

Discussion and conclusions

Our gelatin–albumin based tissue embedding matrix provides a simple method for simultaneous histological processing of multiple tissue samples. It provides several advantages compared to methods that separately process individual samples. First, the amount of labor involved in sectioning, histological processing and subsequent mounting of the samples is reduced. Second, the matrix surrounding the tissue provides support and protection for the tissue during sectioning, thus reducing damage to

Acknowledgements

We are thankful to Dr. Cecilia Hedin-Pereira (Federal University of Rio de Janeiro) for sharing an earlier gelatin–albumin embedding protocol, to Dr. Andrew J. Dwork (New York Psychiatric Institute) for contributing human cerebral cortex, and to Drs. David Dunlop and Amos Neidle (Nathan Kline Institute, New York) for their helpful discussions and reading the manuscript. Supported by NIH grants MH085208, MH64168 and DC04318.

References (6)

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