An Immunolabeling Technique to Visualize the Subcellular Localization of Proteins

Overview

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) for another 2 min. Using gravity pressure, use a 25 G 5/8" needle to introduce the fixatives into the heart. Immediately after the heart starts to fill up with the fixative, lift up the apex of the heart and cut the vessels underneath at 1-2 mm from the heart to relieve pressure and allow the liquids to drain.
3. Dissect the heart, remove the atria, and drop the ventricles into ice-cold 3% glutaraldehyde/0.1 M sodium cacodylate in a Petri dish. Make a butterfly cut after 30-60 min of fixation and place it in a Petri dish containing 3% glutaraldehyde/cacodylate (see Table of Materials). Chop the heart using a surgical blade in small cubes of 1 mm³ while it is in glutaraldehyde/cacodylate solution.
4. Fix/immerse the dissected heart tissue in glutaraldehyde/cacodylate solution for 24 h at 4 °C.

2. Heart tissue processing

1. After 24 h of fixation, wash the tissues in 0.1 M of sodium cacodylate buffer for 20 min.
   1. Prepare 0.1 M cacodylate buffer following the steps below.
      1. To prepare 0.1 M sodium cacodylate buffer, prepare a stock of 1 M sodium cacodylate buffer by dissolving sodium cacodylate (21.4 g) and 1% calcium chloride solution (3 mL) in 90 mL of distilled water. Volume up the solution to 100 mL by adding the distilled water. Stir the solution well and leave it overnight to dissolve the solute.
      2. Next, take 10 mL of 1 M sodium cacodylate stock solution and add it to 80 mL of distilled water. Adjust the pH to 7.4 using HCl and volume it up to 100 mL by adding distilled water.
2. Repeat the wash in 0.1 M sodium cacodylate buffer for another 20 min. Remove the tissues from sodium cacodylate buffer and immerse them in 2% osmium tetroxide solution for 4 h at room temperature. This process is called osmication.
   1. Prepare 2% Osmium tetroxide solution following the steps below.
      1. To prepare 2% Osmium tetroxide solution, take 4% Osmium tetroxide solution, 4 mL (see Table of Materials), 1 M sodium cacodylate stock solution (0.8 mL), and distilled water (3.2 mL) to make a total of 8 mL solution.
         NOTE: This whole process and the steps from hereon must be carried out in the fume hood.
3. After osmication, immerse the tissues in 2% sodium acetate solution for 10 min at room temperature.
   1. Prepare 2% sodium acetate solution by dissolving 4 g of sodium acetate in 20 mL of distilled water.
4. Next, immerse the tissues in 2% uranyl acetate solution for 1 h at room temperature.
   1. Prepare 2% uranyl acetate solution by dissolving 4 g of uranyl acetate in 20 mL of distilled water.
5. After uranyl acetate staining, dehydrate the tissues sequentially through the graded alcohols and acetone in the order mentioned in Table 2.

3. Heart tissue embedding

1. Embed the dehydrated tissues in low-viscosity epoxy resin following the steps below.
   1. To make low-viscosity epoxy resin, mix 10.24 mL of vinyl cyclohexene dioxide epoxy monomer, 6.74 mL of diglycidyl ether of polypropylene glycol (DER 732), and 30.05 mL of nonenyl succinic anhydride (NSA) (see Table of Materials) in a 50 mL tube. Stir the suspension well by hand for 2 min.
   2. Add 18 drops of 2-dimethylaminoethanol (DMAE, see Table of Materials) epoxy accelerator and stir the suspension to mix the components thoroughly.
   3. Impregnate the tissues in epoxy resin in the following sequence of mixture.
      1. Replace the 100% acetone with 1:1 resin: acetone suspension and immerse the tissues in it for 1 h at room temperature.
      2. Replace 1:1 resin: acetone suspension with 6:1 resin: acetone suspension and immerse the tissues in it for 3 h.
      3. Finally, replace the 6:1 resin: acetone suspension with 100% resin suspension and leave tissues immersed in it overnight at room temperature.
2. Put the tissues into fresh resin in 8 mm micro molds (see Table of Materials) and cure the embedded tissues at 70 °C overnight.
   NOTE: Ensure that the resin is hard but not brittle after curing.

4. Tissue sectioning using an ultramicrotome

1. Trim the resin blocks with the tissue to no larger than 1 mm before mounting on the ultramicrotome (see Table of Materials). Place the mold as precisely as possible in the segmented arm of the ultramicrotome and manually advance the sample mold toward the diamond knife.
2. Cut the sections at a thickness of 500 nm (one-half micron) with a Histo knife (see Table of Materials), and using the EM loop tool, pick them up and place them on a glass slide.
3. Place the glass slide on a hot plate for toluidine blue stain to find the area of interest.
4. After finding the area of interest, use an Ultra 45° knife (see Table of Materials) to produce pale gold ultrathin sections (100 nm). Place these ultrathin sections on the dull side of a 200 mesh copper grid.

5. Ultrathin heart section staining

1. Start the staining by unmasking the antigen using an etching solution, i.e., 5% sodium meta-periodate solution.
   1. Prepare 500 µL of 5% sodium metaperiodate (see Table of Materials) solution in distilled water.
      NOTE: Prepare fresh solution before use.
   2. Put 20 µL of sodium metaperiodate solution on a clean paraffin film. Place the completely dried grids with tissue sections on the droplet of the etching solution.
      NOTE: The dull side of the grid with the tissue section must face toward the etching solution.
   3. Leave the section grid on the solution for 30 min at room temperature.
2. Wash the etched tissue section by placing it on a droplet of distilled water for 60 s.
3. Block the residual aldehydes by placing the section grids on a droplet of 0.05 M glycine solution for 10 min at room temperature. Blot the grid's edges on filter paper to remove the residual glycine solution.
   1. Prepare 0.05 M glycine solution (see Table of Materials) by dissolving 3.75 mg of glycine in 1 mL of 1x PBS (pH 7.4).
4. Place the section grids in 10-20 µL of blocking solution for 25 min at room temperature.
   NOTE: Blocking solution composition: 2 µL of 1% normal goat serum (NGS) + 20 µL of 1% BSA (final concentration: 10%) + 178 µL of 1x PBS (pH 7.4) to make a final volume of 200 µL.
5. Blot the grid edges on filter paper and place the grid section on antibody diluent for conditioning at room temperature for 10 min.
6. Incubate the grid sections with primary antibody (Sigmar1 in this case, see Table of Materials; diluted 1:10 in antibody diluent) for 1 h 30 min in a humidified chamber.
7. Blot dry the grid and wash the grid sections with antibody diluent twice for 5 min each.
8. Incubate the grid sections with biotinylated secondary antibody (in this case, biotinylated goat anti-rabbit polyclonal secondary antibody, see Table of Materials; diluted 1:10 in antibody diluent) for 1 h in a humidified chamber.
9. Blot dry the grid and wash the grid sections with antibody diluent twice for 5 min each.
10. Incubate the grid sections in commercially available streptavidin-conjugated QD (QD_655nm, see Table of Materials; diluted 1:10 in antibody diluent) for 1 h in a humidified chamber at room temperature. Prevent exposure to light by covering the chamber with aluminum foil.
11. Blot dry the grid edges using filter paper and wash the grid sections by placing them on water droplets for 2 min.
12. Blot the edges of the grid to dry.

Table 1: Composition of PBS and other solutions used for tissue dehydration.

Buffer	Composition	Quantity
Phosphate-buffered Saline (PBS) (1x) (1 L)	Sodium Chloride	8 g
	Potassium Chloride	0.2 g
	Sodium Phosphate dibasic	1.44 g
	Potassium Phosphate monobasic	0.24 g
	Adjust the pH to 7.4

Table 2: Application of graded alcohol and acetone for tissue dehydration (Step 2.5)

Component	Time
50% ethanol	10 min
70% ethanol	10 min
95% ethanol	10 min
100% ethanol	20 min
100% ethanol	20 min
100% acetone	30 min
100% acetone	30 min

Materials

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