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1Cognitive Neuroscience Unit, Montreal Neurological Institute, 2Ècole d’Optomètrie, Universitè de Montrèal, 3Department of Psychology, McGill University
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Large-scale immunodetection of target proteins across the entire primate brain is possible by employing novel tissue embedding and sectioning methods combined with the use of creative apparatus for batch staining of multiple free-floating sections at a given time.
Zangenehpour, S., Burke, M. W., Chaudhuri, A., Ptito, M. Batch Immunostaining for Large-Scale Protein Detection in the Whole Monkey Brain. J. Vis. Exp. (29), e1286, doi:10.3791/1286 (2009).
Immunohistochemisty is one of the most widely used techniques for characterizing protein expression in the brain of various experimental animal models. It is relatively easy to conduct systematic immunohistochemical procedures on the brains of rodents and other common experimental models with similar brain size. However, there is no published work to our knowledge that provides a comprehensive account of how to carry out such immunodetection procedures across an entire monkey brain. What follows is a detailed description of how to prepare a whole monkey brain for large-scale immunohistochemical detection of various target proteins. This work has emerged as a result of collaboration between commercial and academic endeavours. As such, details pertaining to tissue embedding and sectioning remain a proprietary knowledge of NSA.
Part 1: Animal treatment and tissue preparation
The brain from an adult vervet monkey (Cercopithecus aethiops) is used for the present protocol. All procedures are carried out in compliance with the Canadian Council on Animal Care (CCAC) guidelines for the use and care of animals in biomedical research1.
Part 2: Histological processing
Sections are chosen at a given spatial interval (e.g., 500-µm) and for our experimental purposes were processed with the following antibodies: fragile-X mental retardation protein (FMRP; Chemicon; Temecula, CA), SMI32 (Sternberger Monoclonals Inc.; Baltimore, MD), and NeuN (Chemicon; Temecula, CA). FMRP is a cytoplasmic protein that abundantly found in neurons of normal and permutation carrier brains2. NeuN (Neuronal Nuclei) specifically recognizes the DNA-binding neuron-specific protein NeuN which is present in most neurons and is distributed in neuronal nuclei, perikarya and some proximal neuronal processes3. SMI-32 is a monoclonal antibody that recognizes the non-phosphorylated epitope on neurofilament proteins4.
Part 3: Representative Results:
This method produces a complete expression profile of a target protein of interest across an entire monkey brain. Here we show representative coronal sections that provide a snapshot of FMRP, NeuN and SMI32 expression in the same monkey brain.
Figure 1: Staining dish (A) and basket (B) used for large-scale batch processing of free-floating sections for immunodetection.
Figure 2: Representative coronal sections from a vervet monkey brain stained for FMRP (A), NeuN (B) and SMI32 (C) antibodies.
There are two critical steps in this procedure that make large-scale detection of proteins in an entire monkey brain possible. One is the embedding and sectioning protocol, which remains the proprietary knowledge of NSA. The other is the use of staining dishes and baskets provided by HistoTools. The latter allows for easy and quick handling of many (~40) sections at a given time. It also provides the means for uniformly treating all sections across the brain and makes for a scientifically sound histological treatment. In addition, the use of embedded landmarks provides the added advantage of being able to digitally acquire the staining patterns from dried and coverslipped slides and to subject the digitized files to various forms of analyses. Although we provide examples of three antibodies, this procedure can be applied to a wide range of other target proteins as well as histological stains, particularly those that reveal the cytoarchitecture various cortical areas and hence used for mapping purposes.
The authors have nothing to disclose.
We are grateful to Frank Ervin, Roberta Palmour and the staff of the Behavioural Sciences Foundation Laboratories located in St Kitts, West Indies, for their continued support of our primate work. This work was supported by a fellowship from Fragile X Research Foundation of Canada (FXRFC) to SZ and - operating grants from the Canadian Institutes of Health Research (AC) and the National Engineering and Research Council of Canada (MP).
|anti-FMRP monoclonal antibody||Chemicon International||MAB2160||Requires antigen retrieval in fixed tissue.|
|anti-NeuN monoclonal antibody||Chemicon International||MAB377||N/A|
|anti-Neurofilament H monoclonal antibody||Sternberger Monoclonals Inc.||SMI32||N/A|
|Antigen Unmasking Solution||Vector Laboratories||H-3300||N/A|
|Normal horse serum||Invitrogen||16050122||500 mL|
|Biotinylated Anti-Mouse IgG (H+L), made in horse||Vector Laboratories||BA-2000|
|VECTASTAIN ABC Kit (Standard)||Vector Laboratories||PK-4000||1.5 mg|
|Staining dish for floating sections||HistoTools||10009||12 x 65 mm|
|Staining transport basket||HistoTools||10012||large
(fits 12 x 65 mm dish)
|Triton X-100||Fisher Scientific||P8514B||N/A|
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