Determining the subcellular localization of a protein is critical to delineating its proper function and the mechanisms involved in it. This method can help us visualize the subcellular localization of a protein at the ultra structure level. Quantum dots-mediated immunolabeling is more stable, resistant to photobleaching, has its own emission spectra, yields high electron density, and displays higher efficiency and retention on tissues with better penetration in tissues.
Multiple steps of the process, that is fixation, etching, blocking, and optimal quantum dots concentration, are challenging steps to optimize. It is advisable to try multiple time points and concentrations of the reagents used. Another challenge is handling grids for which only patience and, of course, practice works.
Demonstrating the procedure will be Chowdhury Abdullah, a postal fellow, Naznin Remex, a graduate student from my laboratory, and Brandon Hartman, electron microscopy supervisor from our electron microscopy services. After anesthetizing the mice and opening the chest cavity, profuse the heart using ice cold 3%glutaraldehyde in 0.1 molar of sodium cacodylate buffer for two minutes. Use a 25 gauge needle of five by eight inch to introduce the fixatives into the heart using gravity pressure.
Immediately after the heart starts to fill up with the fixative, lift the apex of the heart to relieve pressure. Cut the vessels underneath at one to two millimeters from the heart and allow the liquids to drain. Dissect the heart.
Remove the atria and drop the ventricles into a Petri dish containing ice cold 3%glutaraldehyde and 0.1 molar sodium cacodylate. Make a butterfly cut after 30 to 60 minutes of fixation and place the ventricle back in the Petri dish. Chop the heart using a surgical blade in small cubes of one cubic millimeter.
Fix and immerse the dissected heart tissue in glutaraldehyde and cacodylate solution for 24 hours at four degrees Celsius. After 24 hours of fixation, wash the tissue in 0.1 molar sodium cacodylate buffer twice for 20 minutes. Remove the tissue from sodium cacodylate buffer and immerse in 2%osmium tetroxide solution for four hours at room temperature.
After osmication, immerse the tissue in 2%sodium acetate solution for 10 minutes at room temperature. Next, immerse the tissue in 2%uranyl acetate solution for one hour at room temperature. After uranyl acetate staining, dehydrate the tissue sequentially through the graded alcohols and acetone.
Embed the dehydrated tissue in low viscosity epoxy resin as described in the manuscript. Put the tissue into fresh resin in eight millimeter micro molds and cure the embedded tissue at 70 degrees Celsius overnight. After finding the area of interest, using an ultra 45 degrees knife, produce pale gold ultrathin sections.
Place these ultrathin sections on the dull side of a 200 mesh copper grid. Start the staining by unmasking the antigen by putting 20 microliters of etching solution metaperiodate on a clean paraffin film. Place the dried grid with tissue sections on the droplet of the etching solution.
Leave the section grid on the solution for 30 minutes at room temperature. Wash the etched tissue sections by placing them on a droplet of distilled water for 60 seconds. Block the residual aldehydes by placing the section grid on a droplet of 0.05 molar glycine solution for 10 minutes at room temperature.
Blot the grid's edges on filter paper to remove the residual glycine solution. Place the section grid in 10 to 20 microliters of blocking solution for 25 minutes at room temperature. Blot the grid edges on filter paper and place the grid sections on antibody diluent for conditioning at room temperature for 10 minutes.
Incubate the grid sections with primary antibody for one hour 30 minutes in a humidified chamber. Blot dry the grid and wash the grid sections with antibody diluent twice for five minutes each. Incubate the grid sections with biotinylated secondary antibody for one hour in a humidified chamber.
Once the grid is blot dried, wash the grid sections with antibody diluent twice for five minutes each. Incubate the grid sections in commercially available streptavidin conjugated QD for one hour in a chamber at room temperature. Prevent exposure to light by covering the chamber with aluminum foil.
After blot drying the grid edges using filter paper, wash the grid sections by placing them on water droplets for two minutes and blot the grid edges to dry. Insert the specimen holder in the microscope column and engage the pump switch to evaluate the goniometer, followed by full insertion of the specimen holder in the microscope column. Focus well on the desired area.
Capture the image using a high-speed digital camera and save the file in tif format. The mitochondrial membranes, lysosomes, and the endoplasmic reticulum or sarcoplasmic reticulum membrane mitochondrial interface showed the presence of Sigmar1 labeled quantum dots. Heart sections were visualized using rabbit immunoglobulin G and quantum dots as an isotype control for anti-Sigmar1 rabbit primary antibody.
One important thing to consider while working on this protocol is unmasking time for antigen. If the tissue sections are etched for too long, the metaperiodate solution will create perforations in thin tissue sections. Quantum dot-mediated labeling is gaining attention in tumor detection, tumor molecular and immune status profiling, and tissue imaging paving a new way for medical diagnostics.
Quantum dots are also useful in treating tumors via photodynamic therapies and ophthalmic anomalies by delivering medicine to the eyes.
