An Immunolabeling Technique to Visualize the Subcellular Localization of Proteins

Abstract

Source: Aishwarya, R., et al. Visualizing Subcellular Localization of a Protein in the Heart Using Quantum Dots-Mediated Immuno-Labeling Followed by Transmission Electron Microscopy. J. Vis. Exp. (2022).

This video demonstrates a technique for the immunolabeling of sigma-1 receptors in a murine heart tissue section using quantum dots. The heart tissue is fixed, osmicated, stained, dehydrated, embedded in resin, and then sectioned. The sections are then treated with antibodies and labeled with quantum dots to demarcate the subcellular localization of Sigma-1 receptors using electron microscopy.

Protocol

1. Animal preparation

1. Anesthetize the mice using 3% isoflurane. Open the chest by making a horizontal incision in the region above the middle abdomen and pulling the skin. Carefully make another incision to open the chest cavity without puncturing any other organs.
   1. Perfuse the heart through the apex first and then the right ventricle using cold 3% glutaraldehyde in cardioplegic solution (50 mM KCl, 5% dextrose in PBS, see Table 1) for 2 min to ensure complete myocardial relaxation.
2. Perfuse the heart using ice-cold 3% glutaraldehyde in 0.1 M of sodium cacodylate buffer (pH 7.4) ....

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
200 Mesh copper grid	Ted Pella	G200HH
6-month-old male mice with FVB/N background	Jackson Laboratory, Bar Harbor, ME
Acetone 100%	Fisher Chemicals	A949
Antibody diluent	Dako	S3022
anti-Sigmar1 antibody	Cell Signaling	61994S
Biotinylated goat anti-rabbit IgG antibody	Sigma Aldrich	B7389
BSAc (10%)	Electron Microscopy Sciences	25557
Calcium chloride	Sigma Aldrich	C7902
Cytoseal Xyl	Thermo fisher	8312-4
DER 732C36AA10:C33	Electron Microscopy Sciences	13010
Dextrose	Sigma Aldrich	G7528
Diamond Knife	Diatome	Histo; Ultra 450
DMAE	Electron Microscopy Sciences	13300
Electron microscope	JEOL	JEOL-1400 Flash
ERL 4221	Electron Microscopy Sciences	15004
Ethanol 100%	Fisher Chemicals	A405P
Glutaraldehyde 3%	Electron Microscopy Sciences	16538-15
Glycine	Alfa Aesar	A13816
Hydrochloric acid	Fisher Scientific	SA56
Micromolds	Ted Pella	10505
Microtome	Leica Microsystem	EM UC7
Normal goat serum	Invitrogen	PCN5000
NSA	Electron Microscopy Sciences	19050
Osmium tetroxide	Electron Microscopy Sciences	19150
Parafilm	Genesse Scientific	16-101
Potassium Chloride	Sigma Aldrich	P5655
Potassium Phosphate monobasic	Sigma Aldrich	71640
Qdot 655 Streptavidin Conjugate	Invitrogen	Q10121MP
Sodium Acetate	Fisher Scientific	BP334
Sodium Cacodylate	Electron Microscopy Sciences	12300
Sodium Chloride	Fisher Scientific	BP358
Sodium metaperiodate	Sigma Aldrich	71859
Sodium Phosphate dibasic	Sigma Aldrich	P9541
Surgical blade (size 10)	Aspen surgical	371110
TEM image software	AMT-V700	AMT TEM imaging systems
TEM imaging camera	XR80 TEM series	AMT TEM imaging systems
Toluidine Blue O solution (0.5%)	Fisher Scientic	S25612
Uranyl acetate	Polysciences	21447