Batch Immunostaining for Large-Scale Protein Detection in the Whole Monkey Brain

Summary

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.

Abstract

Immunohistochemistry (IHC) is one of the most widely used laboratory techniques for the detection of target proteins in situ. Questions concerning the expression pattern of a target protein across the entire brain are relatively easy to answer when using IHC in small brains, such as those of rodents. However, answering the same questions in large and convoluted brains, such as those of primates presents a number of challenges. Here we present a systematic approach for immunodetection of target proteins in an adult monkey brain. This approach relies on the tissue embedding and sectioning methodology of NeuroScience Associates (NSA) as well as tools developed specifically for batch-staining of free-floating sections. It results in uniform staining of a set of sections which, at a particular interval, represents the entire brain. The resulting stained sections can be subjected to a wide variety of analytical procedures in order to measure protein levels, the population of neurons expressing a certain protein.

Protocol

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....

Discussion

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.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
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
DAB	Fisher Scientific	AC11209-0050	N/A