Immunolabeling of Neuronal Membrane Proteins in a Freeze-fractured Specimen of Mouse Brain Tissue

Abstract

Source: Schönherr, S., et. al. Combined Optogenetic and Freeze-fracture Replica Immunolabeling to Examine Input-specific Arrangement of Glutamate Receptors in the Mouse Amygdala. J. Vis. Exp. (2016)

This video demonstrates the immunolabelling of freeze-fractured replicas of mouse brain tissue for electron microscopy. The fractured and coated replicas are digested to expose receptors. Primary and gold-tagged secondary antibodies are added to label specific neuronal membrane receptors. The replica is mounted on a grid and visualized under an electron microscope.

Protocol

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

1. Specimen Preparation

1. Brain fixation
   1. Preparation of the fixative
      1. For 1 L of fixative, weigh 10 g of paraformaldehyde and add it to 300 ml of deionized water (H₂O). Heat to 55 - 60 ºC for ~10 min with continuous stirring.
      2. Switch off the heat and add 7 - 8 drops of 4 N Sodium Hydroxide (NaOH). The solution should become clear in ~10 minutes.
      3. Let it cool down to room temperature or RT, add 150 ml ....

Results

Figure 1. Tissue Preparation and High-pressure Freezing. (A) Vibroslicer is used to sec.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Tissue preparation
Paraformaldehyde EM grade	Agar Scientific Ltd., United Kingdom	AGR1018
Saturated picric acid solution	Sigma-Aldrich, USA	P6744-1GA
Na2HPO4-2H20	Merck Millipore, Germany	1065860500
NaH2PO4-2H2O	Merck Millipore, Germany	1063451000
NaCl	Merck Millipore, Germany	1064041000
4N NaOH	Carl Roth, Germany	T198.1
Thiopental	Sandoz, Austria	5,133
Glycerol	Sigma-Aldrich, USA	G5516-500ML
GenPure ultrapure water system	Thermo Fisher Scientific, USA	50131235
Peristaltic pump	ISMATEC, Germany	ISM 930C
Filter Paper	MACHEREY-NAGEL, Germany	MN 615 1/4
Vibroslicer, VT1000S	Leica Microsystems, Austria
Ophthalmic scalpel	Alcon Laboratories, USA
Perfusion cannula	Vieweg, Germany	F560088-1
High-pressure Freezing
Copper carriers	Engineering Office M. Wohlwend, CH	528
Sidol Polish	Henkel, Germany
Chamois skin	Household supply store
Hole punch, 1,5mm	Stubai, Austria
Denatured ethanol	Donauchem, Austria
Acetone	Roth, Germany	9372.5
High Pressure Freezing Machine HPM 010	BalTec, CH; now Leica Microsystems	HPM010
Stereo-microscope	Olympus, Japan	SZX10
Liquid nitrogen
Cryo-vials	Roth, Germany	E309.1
CryoCane	Nalge Nunc International,USA	5015-0001
CryoSleeve	Nalge Nunc International,USA	5016-0001
Liquid nitrogen storage vessel	Cryopal, France	GT38
Non-ionic detergent (Lavocid)	Werner & Mertz Professional, Germany
Freeze-fracture and Replication
Sandblaster, Mikromat 200-1	JOKE Joisten & Kettenbaum, Germany	SANDURET 2-K
Siliciumcarbid SIC 360, grain size 25 - 21µ	JOKE Joisten & Kettenbaum, Germany	955932
Freeze Fracture System BAF 060	BalTec, CH; now Leica Microsystems	BAF060
Ceramic 12 well plate	Gröpel, Austria	14511
Trizma base	SIGMA, USA	T1503
Trizma hydrochloride	SIGMA, USA	T3253
Sodium chloride	Merck, Germany	1,06,40,41,000
SDS, Sodium lauryl sulfate	Roth, Germany	5136.1
Sucrose	Merck, Germany	1,07,68,71,000
TRIS	Roth, Germany	5429.3
Universal Hybridization Oven	Binder, Germany	7001-0050
Immunolabelling
BSA	SIGMA, USA	A9647
Anti-GFP Antibody	Molecular Probes, USA	A11122
Anti-pan-AMPAR Antibody	Frontier Institute, Japan	pan AMPAR-GP-Af580-1
Anti-NMDAR1 Antibody, clone 54.1	Merck Millipore, Germany	MAB363
Opioid Receptor-Mu (MOR) Antibody	ImmunoStar, USA	24216
EM goat anti-guinea pig, 5nm; secondary antibody	BBInternational,	EM.GAG5
EM goat anti-rabbit, 15nm; secondary antibody	BBInternational,	EM.GAR15
Donkey anti-rabbit, 10nm, secondary antibody	AURION, Netherlands	DAR 10nm
Copper grids, 100 Parallel Bar	Agar scientific, UK	G2012C
Incubator	Major Science, USA	MO-RC
Pioloform Powder	Agar scientific, UK	R1275
Chloroform	Roth, Germany	3313.1