Basic neuroscienceAn improved approach to align and embed multiple brain samples in a gelatin-based matrix for simultaneous histological processing
Graphical abstract
Introduction
Histological analysis of biological tissue is essential for studying cellular morphology and phenotypes in situ. However, the process to prepare microscope-ready tissue samples is rather laborious, and more time and effort are required when a large number of tissue samples needs to be analyzed. In clinical pathology laboratories, tissue microarray, a method to punch out pieces of tissues and embed them in paraffin in an ordered arrangement, has been developed and is often utilized for simultaneous analysis of multiple biopsied specimens (Kononen et al., 1998, Miettinen, 2012). In basic biomedical research, where the analysis of whole organ tissues from experimental animals may be preferred, tissue embedding techniques have been more commonly used to protect the structure of complex organs such as the brain (Bjarkam et al., 2001, Griffioen et al., 1992) and cochlea (Hurley et al., 2003) during processing rather than to process multiple samples at the same time.
Recently, Smiley and Bleiwas (2012) have described a method to embed multiple mouse brains. In their protocol, the brain samples were secured on a solid surface with pins and embedded in a gelatin-based matrix containing several ingredients including albumin, lysine and glutaraldehyde. While they successfully demonstrated simultaneous sectioning and staining of multiple brain samples, the method requires each brain sample to have an extra tissue area (e.g., brainstem) to be pinned or risk damaging a portion of the sample by the pinning process. This requirement poses a challenge when smaller samples such as neonatal or half-brain samples must be secured. Furthermore the inclusion of additional constituents to facilitate cross-linking of gelatin to the tissue samples makes the preparation of the matrix more complicated and costly.
In this report, we describe a new method to effortlessly align and embed multiple brain samples in a gelatin-based matrix block for simultaneous tissue processing. With two prototypes of specifically designed brain array casts, we first created a mold or sample receiving matrix with sample-shaped cavities in an organized arrangement. Using this technique, we were able to eliminate the pinning process and align small, mouse brain hemispheres without damaging any part of the tissue. The gelatin block with embedded samples can be ready for frozen sectioning within 34 days without many of the additional ingredients included in previously described embedding matrices (Smiley and Bleiwas, 2012).
Section snippets
Materials
Gelatin (from porcine skin, type A, gel strength 300) was obtained from Sigma-Aldrich Co. (St. Louis, MO, USA) and used to make a gelatin-based embedding matrix. Brain array casts were specifically designed in order to align multiple mouse brains and embed them in a single block. Two prototypes tested in this study were constructed with high heat-sensitive polymer (high-temperature melting glue gun glue) and liquid rubber (Fig. 1). The rabbit polyclonal anti-mouse Iba-1 antibody was purchased
Brain array cast prototypes efficiently created sample receiving matrices with multiple close-fitting brain-shaped cavities that securely held brain tissue
To develop a technique to simplify aligning and embedding of multiple brain tissue samples, two designs of brain array cast prototypes were tested (Fig. 1A and B). Both prototypes produced functional sample receiving matrix, providing well-aligned, brain-shaped impressions (Fig. 2A-2 and B-2). By comparison the removal of the sample receiving matrix was easier with the liquidrubber-based brain array cast. The polymer-based brain array cast had a higher tendency to adhere to the gelatin matrix
Discussions and conclusions
Two prototypes of brain array casts that differed in the materials (liquid rubber vs. heat-sensitive polymer) and designs (built-in container vs. casting insert plus container) were tested (Fig. 1A and B). Both designs successfully produced effective sample receiving matrices with brain-shaped cavities that securely held multiple brain samples in a vertical position during embedding. Removal of sample receiving matrix from the cast constructed from liquid rubber required less effort than that
Acknowledgements
This work was supported by the Department of Pathology, University of North Dakota School of Medicine and Health Sciences and NIH grant number 5R01AG042819.
References (7)
- et al.
New strategies for embedding, orientation and sectioning of small brain specimens enable direct correlation to MR-images, brain atlases, or use of unbiased stereology
J Neurosci Methods
(2001) - et al.
Gelatin embedding to preserve lesion-damaged hypothalami and intracerebroventricular grafts for vibratome slicing and immunocytochemistry
J Neurosci Methods
(1992) - et al.
Cochlear immunochemistrya new technique based on gelatin embedding
J Neurosci Methods
(2003)
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