Protocols

The Gel Monomer Stock Solution is made to a final concentration of 1x PBS and can be made in advance and stored at 4 ˚C for up to one month.

Final Gel Solution is made immediately prior to application onto tissue. N,N,N′,N′-Tetramethylethylenediamine and Potassium Persulfate are added last to prevent premature gelling.

Allow the Final Gel Solution to diffuse into the tissue for 30 minutes at 4 ˚C. Place a glass slide on top of the gel to prevent exposure to air. Polymerization can be completed in as few as 3 hours (2 hours at 37-45 ˚C, 1 hour at 60 ˚C) or can be left overnight at 37-45˚ C in a humidified container (we use a petri dish with a wet paper towel).

Homogenization solution may be stored at room temperature. It is advisable to measure pH before use. Alternatively, TRIS buffer may be added as needed to prevent pH drift.

 Homogenization Time by Tissue Type

Antibody Staining

Wash expanded samples 3 times for 10 minutes each in 1× PBS at RT. An optional blocking step may be performed (3% bovine serum albumin in 0.1% Triton-X100 in 1x PBS works well for us) before incubating with primary antibodies in Staining Buffer overnight.

Wash samples three times for at least 10 minutes each in 1× PBS at RT before incubation in the corresponding secondary antibodies for 3 hours in the same conditions as the primaries. Prior to imaging, wash with 1× PBS, and optionally expand further with dilute PBS (1:10 or 1:50 dilution in ddH2O) or in pure ddH2O.

NHS Ester Staining

Shrink the gel in polyethylene glycol (PEG 200) before staining with NHS-Ester (our favorites are NHS-ATTO-488, NHS-ATTO-532, and NHS-Cy3) diluted to 1:25-1:150 for 3 hours at RT in staining buffer (1× PBS, 2× SSC, and 100 mM sodium bicarbonate solution all give similar results).

Lipid Staining

For lipid visualization, stain with DiO, DiI, or DiD diluted to 1:200 for 72-96 hours in 0.1% TritonX-100 at RT. Wash at least three times with 1× PBS before optional washing in water until the sample is fully expanded, at least three exchanges of water for 10 minutes each. Fully expanded samples stained with lipophilic dyes should be imaged as close to the time of expansion as possible to prevent dissociation of the dye in water.

FISH

For Magnify samples being processed for DNA FISH probing, place homogenized gel samples in pre-heated hybridization buffer made of 2× SSC (300 mM NaCl, 30 mM sodium citrate, pH 7.0) 10% (v/v) dextran sulfate, 20% (v/v) ethylene carbonate, 0.1% (v/v) Tween20 at 60 °C  for 30 min. Incubate the gel samples with hybridization buffer containing 10 pM (per oligo) of FISH probes against target gene (at least 20 probes per 10kb) at 45 °C overnight. Wash twice with stringency wash buffer made of 2× SSC and 0.1% Tween20 at 45 °C for 15 min, followed by two washes with 2× SSCT at 37 °C for 10 min each. Finally, wash with 1× PBS multiple times at RT (5 min each) prior to imaging, or storage at 4°C.

During polymerization, we prefer to cover the gel with another glass slide rather than a coverslip, as we find it easier to remove.

When staining, 6-well plates are generally suitable to hold the gels. For full expansion, washing is generally performed in a petri dish.

When in 1× PBS (~4-5× expanded), gels can generally be easily moved with a wide paint brush. For larger or more fully-expanded gels, a thin, flexible sheet of plastic (for instance, cut from a plastic face shield or large soda bottle) can be used to make gel handling easier, by sliding it under the gel before moving it elsewhere.

To prevent sample drift while imaging, 0.1% poly-L-lysine can be applied to the glass surface before gel transfer. Likewise, to prevent evaporation from the gel while imaging, plastic cling wrap may be applied over the gel.