An Immunolabeling Technique to Visualize the Subcellular Localization of Proteins
Published: April 30, 2024
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 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. 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 com…
開示
The authors have nothing to disclose.
Materials
| 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 |
