Primer for Immunohistochemistry on Cryosectioned Rat Brain Tissue: Example Staining for Microglia and Neurons

Summary

This introductory level protocol describes the reagents, equipment, and techniques required to complete immunohistochemical staining of rodent brains, using markers for microglia and neuronal elements as an example.

Abstract

Immunohistochemistry is a widely used technique for detecting the presence, location, and relative abundance of antigens in situ. This introductory level protocol describes the reagents, equipment, and techniques required to complete immunohistochemical staining of rodent brain tissue, using markers for microglia and neuronal elements as an example. Specifically, this paper is a step-by-step protocol for fluorescent visualization of microglia and neurons via immunohistochemistry for Iba1 and Pan-neuronal, respectively. Fluorescence double-labeling is particularly useful for the localization of multiple proteins within the same sample, providing the opportunity to accurately observe interactions between cell types, receptors, ligands, and/or the extracellular matrix in relation to one another as well as protein co-localization within a single cell. Unlike other visualization techniques, fluorescence immunohistochemistry staining intensity may decrease in the weeks to months following staining, unless appropriate precautions are taken. Despite this limitation, in many applications fluorescence double-labeling is preferred over alternatives such as 3,3'-diaminobenzidine tetrahydrochloride (DAB) or alkaline phosphatase (AP), as fluorescence is more time efficient and allows for more precise differentiation between two or more markers. The discussion includes troubleshooting tips and advice to promote success.

Introduction

Immunohistochemistry is a process for detecting antigens (i.e. proteins) in tissue sections by using primary antibodies which bind specifically to the antigens of interest. Immunohistochemistry was pioneered by J.R. Marrack in 1934 when he determined that antibodies could localize antigens with great specificity¹. Beginning in 1942, some of the first in vitro studies using fluorescent antibodies to visualize immunohistochemistry were published^2,3, after which the first in vivo histochemical study was published ⁴. During the 1960’s, three decades after the inception of immunohistochemical methods, enzyme-conju....

Protocol

NOTE: All procedures were carried out in compliance with The Institutional Animal Care and Use Committee (IACUC) of the University of Arizona. A list of recommended materials and equipment can be found in Table 1.

1. Tissue Preparation

1. Perfusion
   1. Euthanize rodent with an overdose of sodium pentobarbital (25 mg/kg, IP), and perfuse transcardially with phosphate buffered saline (PBS) until completely exsanguinated (3-5 min) at a flow rate of 8 ml/min. For in-depth perfusion instructions, see Gage et al 2012⁹.
   2. Immediately following PBS flush, fix tissue by perfusing with 4% paraform....

Results

This staining protocol results in rat brain tissue sections that have microglia fluorescently labeled in the 594 channel (red) and neurons labeled in the 488 channel (green; see Figure 4). If a nuclear stain has been done, it will show in the 405 channel (blue). Images can be taken in different channels and overlaid for direct comparison of the three channels, or between any two channels. Many digital acquisition software suites include this functionality. Double-labeling with Iba1 and Pan-neuronal marke.......

Discussion

The overall goal of this communication was to introduce immunohistochemistry procedures to the reader. For this, the example of double-labeling with Iba1 and Pan-neuronal antigens to observe microglia and neurons in paraformaldehyde perfused, sucrose cryoprotected, cryosectioned rat brain was used.

This technique can be adapted to serve endless purposes. An array of different antigens in a variety of tissue types such as, but not limited to brain, lung, liver, kidney and intestine can be visua.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Fisherbrand Superfrost Plus Glass Slides	Fisher Scientific	22-034-979	Used for tissue mounting (1.2.2)
Oven	Thermo Scientific	51028112	Used for tissue drying (2.1.1)
Mini Pap pen	Life Technologies	00-8877	Used in step 2.2.2
Andwin Scientific Tissue-tek Slide Staining Dish	Fisher Scientific	22-149-429	Used for all washes during staining (2.2), as well as the Hoechst step (2.2.8)
Kimwipes	Fisher Scientific	06-666-A	Used for drying slides (2.2)
Black Staining Box	Ted Pella	21050	Used for blocking and staining steps (2.2)
Normal Donkey Serum	Fisher Scientific	50-413-253	Used for block and antibody incubation (2.2)
Mouse α-Pan-neuronal	Millipore	MAB2300	Used for primary antibody (2.2.4)
Rabbit α-Iba1	Wako Chemical	019-19741	Used for primary antibody (2.2.4)
Donkey α-rabbit 594	Jackson ImmunoResearch	711-585-152	Used for secondary antibody (2.2.6)
Donkey α-mouse 488	Jackson ImmunoResearch	715-545-150	Used for secondary antibody (2.2.6)
Caterer's foil	Any	N/A	Used in steps 1.2.2 and 2.3.2
Fluoromount-G	Southern Biotech	0100-01	Used for coverslipping (2.2.8)
Coverslips	Fisher Scientific	12544E	Used for coverslipping (2.2.8)
Clear Nail Polish	Any	N/A	Used for coverslipping (2.2.8)
Axio Observer.Z1 and LSM 710 (laser scanning, confocal)	Carl Zeiss	N/A	Used for imaging (3)
Axioskop A2	Carl Zeiss	N/A	Used for imaging (3)
CitriSolv	FisherScientific		For DAB protocol
ABC	Vector Laboratories	PK-6100	For DAB protocol
DAB Peroxidase kit	Vector Laboratories	SK-4100	For DAB protocol
Biotinylated horse α-rabbit IgG	Vector Laboratories	BA-1100	For DAB protocol
Biotinylated horse α-mouse IgG	Vector Laboratories	BA-2001	For DAB protocol
30% Hydrogen Peroxide	FisherScientific	H325-500	For DAB protocol
Wheaton slide racks and staining dishes	TedPella	21043	For DAB protocol
Masterflex perfusion pump and tubing	Cole-Parmer		Used for perfusion (1.1.1 and 1.1.2)
Andwin scientific tissue-tek CRYO-OCT compound (case of 12)	Fisher Scientific	14-373-65	Used for tissue freezing (1.2.1)
Thermometer (-50 to 50 C)	Fisher Scientific	15-059-228	Used for tissue freezing (1.2.1)
Cryostat	Leica	CM3500S	Used for tissue sectioning (1.2.2)
Staining Dish, Plastic with 2 Lids	Grale Scientific	353	For antigen retrival
20 Place Staining Rack, Slides Horizontal	Grale Scientific	354	For antigen retrival
Microwave	Any	N/A	For antigen retrival