To begin, take the digested and expanded archived or fresh clinical tissue sample and proceed with antibody staining by placing it in a single well of a six-well cell culture plate. Wash the sample three times with PBS for 10 minutes each at room temperature. Depending on the sample size, add 0.5 to 2 milliliters of the primary antibodies in the staining buffer to cover the gel adequately.
Incubate overnight at 37 degrees Celsius. Then, wash the sample three times with PBS for 10 minutes each at room temperature. Add the corresponding secondary antibodies and incubate for three hours at 37 degrees Celsius in the staining buffer of choice.
Rewash the sample as demonstrated earlier, and add 1 to 2 milliliters of polyethylene glycol onto the sample in a six-well plate. Stain the sample with Fluor 4 conjugated NHS ester for three hours at room temperature. At the end of the incubation, wash the sample three times with PBS.
Next, to perform lipid staining, take the expanded tissue sample washed three times in PBS, apply a conventional lipophilic dye diluted 200 fold in 2 milliliters of 0.1%Triton X-100 or PBS for 72 to 96 hours at room temperature, and wash at least three times with PBS. To perform FISH, prepare the hybridization buffer by combining 2 x SSC, 10%dextran sulfate, 20%ethylene carbonate, and 0.1%Tween 20. Place homogenized gel samples in a hybridization buffer preheated at 60 degrees Celsius for 30 minutes.
Then, incubate the gel samples with a hybridization buffer containing 10 picomolar FISH probes against the target gene overnight at 45 degrees Celsius. Wash the samples with stringency wash buffer twice for 15 minutes each at 45 degrees Celsius and twice for 10 minutes at 37 degrees Celsius. Wash the sample three times with PBS for 10 minutes and proceed with imaging or storing the sample in PBS plus 0.02%sodium azide at 4 degrees Celsius.
To fully expand and image the tissue, move the samples expanded fourfold in PBS to a glass bottom six-well imaging plate. If the sample has been expanded further, cut it into smaller pieces. Apply 0.1%polylysine to the glass surface.
Place the sample on the slide, and remove any excess liquid around the gel with a paper laboratory cloth to prevent gel movement during imaging. Afterward, perform fluorescence imaging using a conventional wide-field microscope, a confocal microscope, or another imaging system of choice. After imaging, shrink the samples in PBS with at least three 10-minute washes, as long-term storage in deionized water leads to degradation of the fluorescent signal.
Finally, store the samples in PBS with 0.02%sodium azide at 4 degrees Celsius. Confocal images of fully expanded mouse brain tissue allowed the visualization of proteins and cell and mitochondrial membrane structures. The nucleic acids of formal and fixed paraffin-embedded human lymph node tissue were also identified.
After a 3.5-fold expansion of the kidney section, the crack-free expanded glomerulus was seen. However, inadequate anchoring or homogenization resulted in cracking, distortions, and the loss of labeled targets in another kidney section.
