Performing Immunohistochemistry on a Non-Human Primate Brain Tissue

Abstract

Source: Eid, L. & Parent, M. Preparation of Non-human Primate Brain Tissue for Pre-embedding Immunohistochemistry and Electron Microscopy. J. Vis. Exp. (2017).

This video demonstrates the preparation of aldehyde-fixed non-human primate brain tissue using pre-embedding immunohistochemistry for electron microscopy. It outlines the steps for immunolabeling a specific neuronal protein, followed by a peroxidase reaction that produces a colored precipitate, enabling visualization of the target protein in electron microscopy.

Protocol

All procedures involving animal samples have been reviewed and approved by the appropriate animal ethical review committee.

1. Pre-embedding Immunohistochemistry (Figure 1C)

1. Prepare a 4 L stock solution of Tris-buffered Saline (TBS, 50 mM, pH 7.6) as follows.
   1. M....

Results

Figure 1: Schematics of Essential Steps of the Protocol.

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Dibasic anhydrous sodium phosphate	Fisher scientific	S374-500
Monobasic monohydrate sodium phosphate	EM Science	SX0710-1
Sodium chloride (NaCl)	Fisher scientific	S271-3
Hydroxymethyl aminomethane (THAM)	Fisher scientific	T370-500
HCl	EMD	HX0603-3	1N dilution. Product is corrosive. Use with appropriate potection.
NaOH	EMD	SX0590-1	5N dilution. Product is corrosive. Use with appropriate protection.
Paraformaldehyde	Sigma	P6148	4% dilution. Product is highly volatile in its powder form and highly toxic. Use with caution under a venting hood with appropriate protection.
Acrolein (90%)	sigma	110221	3% dilution. Product is highly toxic. Use under a venting hood with appropriate protection.
Autopsy venting table	Mopec	CE400
Electronic perfusion pump	cole parmer	masterflex L/S 7523-90
Needle (perfusion)	terumo	NN-1838R	18G 1 1/2
Needle	terumo	NN-2713R	21G 1/2
Ketamine			20 mg/kg
Xylazine			4 mg/kg
Acepromazine			0.5 mg/kg
Scalpel
Scalpel blades	Feather lance	201011 J9913	No.22 for surgery and No. 11 for EM
Surgical scissors
Rongeurs
Vibratome	Leica	VT 1200S	Calibrate blade before each use, when the device allows it
Vibratome razor blade	Gillette	GIN 642107
Glycerol	Fisher scientific	G33-4	30% dilution
Ethylene glycol	Fisher scientific	E178-4	30% dilution
Sodium borohydride	sigma	S-9125
Normal horse serum	Jackson immunoResearch Laboratories	008-000-121	2% dilution
Cold-fish gelatin	Aurion	900.033	0.5% dilution. Original product is concentrated at 40%
Primary antibody, SERT	Santa Cruz biotechnology	SC-1458	1/500 dilution
Primary antibody, ChAT	Chemicon (Millipore)	AB144P	1/25 dilution
Primary antibody, TH	ImmunoStar	22941	1/1000 dilution
Biotinylated secondary antibody, goat	Vector laboratories	BA-9500	1/1000 dilution
Biotinylated secondary antibody, mouse	Vector laboratories	BA-2000	1/1000 dilution
Vectastain elite ABC kit	Vector laboratories	PK6100	8.8 µL/mL of A and B each
3'3 diaminobenzidine (DAB)	Sigma	D5637	0,05% dilution. Product is highly volatile in its powder form and toxic. Do not throw waste in the sink.
Peroxide 30%	Fisher scientific	H-323	0,005% dilution