One major limitation of expansion microscopy is fluorescence slows after polymerization and digestion. Label retention expansion microscopy, we call it LR-ExM, uses tri functional anchors, which are in order to polymerization and digestion. After we use tri function anchor, we introduce fluorescence after expansion step.
So we keep good signal to noise ratio while having a high resolution. Label retention expansion microscopy can be combined with any other previously introduced expansion microscopies, and also fluorescent microscopies. It is very robust in versatile method.
And for high regulation imaging, it is really important to have a enhanced signal. To begin, culture cells on a 16 well removable chambered cover glass with complete medium at 37 degrees Celsius and 5%carbon dioxide. Once the cell counts reach approximately 40, 000, fix cells with 100 microliters of 3.2%PFA in PEM buffer for 10 minutes at room temperature.
Then permeabilize cells with the 100 microliters of permeablization buffer at room temperature for 15 minutes. Incubate cells with the streptavidin solution diluted in a cell blocking buffer for 15 minutes at room temperature, and then rinse briefly with 200 microliters of cell blocking buffer. Next, incubate cells with 100 microliters of biotin solution diluted in a cell blocking buffer for 15 minutes at room temperature.
Afterwards, incubate cells with 100 microliters of primary antibody solution containing rat anti alpha tubulin antibody and rabbit anti clathrin heavy chain antibody for either 16 hours at four degree Celsius or one hour at room temperature. Then incubate cells with 100 microliters of secondary antibody solution containing tri functional anchors, donkey anti rabbit dig MA, and donkey anti rat biotin MA for one hour at room temperature. Incubate cells with 100 microliters of 0.25%Glutaraldehyde for 15 minutes at room temperature In order to anchor the proteins onto the hydrogel.
Remove the upper structure of the 16 well plate with the razor blade or any removing tool of choice and keep the bottom cover glass. Place the cover glass in a Petri dish and place them on ice. Add 45 microliters a monomer solution to each well to condition cells.
Incubate on ice for five minutes. To make the gelation solution, prepare a 1.5 milliliter micro centrifuge tube. Mix monomer, double distilled water, and 10%T med.
Do not add the ammonium per sulfate yet. Meanwhile, keep the gelation solution tube on the ice. Add 10%ammonium persulfate to the gelation solution.
Immediately pipette 40 microliters of gelation solution onto each well while storing a gelation solution tube on ice. Place the well plate on ice for three more minutes. To keep the humidity of the gel during the 37 degree Celsius incubation, covered the Petri dish with the lid and put a few drops of water in the Petri dish.
While protecting the sample from light, move the cover glass and the 10 centimeter Petri dish to a 37 degree Celsius incubator for 1.5 hours for gelation. After gelation, cut the cover glass to separate each gel. Transfer gels to six well plates.
Add two milliliters of digestion buffer to each well. Leave the samples either overnight at room temperature or for four hours at 37 degrees Celsius. Then wash gels with at least 10 fold excess volume of water than the final gel volume while repeating the washing step four times for 20 to 30 minutes each time, and the gel expands by about four folds in each dimension.
Incubate gels in two milliliters volume of streptavidin digoxigenin staining buffer with two to five micromolar streptavidin dye and/or anti digoxigenin dye for 24 hours at room temperature while keeping the samples in the dark. Then wash and expand the gel two to four times with excessive water. Each wash takes about 30 minutes to one hour.
For easier visualization of cells under fluorescent microscopy, stain cells with DAPI during the third wash. Dilute DAPI stock in one to 5, 000 delusions in wash water. Incubate the gel with a DAPI solution for 30 minutes to one hour.
Then wash two additional times with three milliliters of water. Coat the six well glass bottom imaging chamber with three milliliters of 0.01%polylysine to immobilize the gel samples. Then transfer gel samples to the coded imaging chamber and image the samples with any desired fluorescence scopes.
Label retention expansion microscopy shows enhanced fluorescence labeling compared to protein retention expansion microscopy or biotin expansion microscopy samples. Label retention expansion microscopy shows about six times higher fluorescence signal compared to protein retention expansion microscopy. LR-ExM can effectively capture small structures, such as clathrin coated pits with subdefraction limit resolution.
Microtubules and clathrin coated pits were co-immuno stains using functional anchors, NHS MA dig, and NHS MA biotin. Similarly, clathrin coated pits and mitochondria were labeled using an enzymatic protein tag approach with tri functional anchors, such as BG MA biotin and BC MA dig. Furthermore, immuno staining based approach and the enzymatic protein tag approach are combined to show the structure of lamin A/C and nucleopore complex.
Label retention expansion microscopy was performed on the mouse brain tissue by co-immuno staining the presynaptic marker, Bassoon, and postsynaptic marker, Homer 1. The two labels were clear and well separated, which supports high-resolution and labeling efficiency. Labor retention expansion microscopy is a versatile and robust method, which can be combined with any other previously introduced expansion microscopies.
For high resolution imaging, it is important to achieve a good labeling efficiency to better capture the molecular scale structure. Labor retention expansion microscopy is effective and robust method to enhance the signal.
