Gel embedding protocol

adapted from Moffitt et al., PNAS 2016, "High-performance multiplexed..."

Materials

Wash 40-mm-diameter #1.5 coverslips for 30 min via immersion in:
- 1:1 mix of 37%(vol/vol) HCl and methanol at room temperature (RT).
Rinse 3x in DI H2O
Rinse 1x in 70% Ethanol
dry at 70C
in Fume-hood: for 30 min at RT, immerse in silanization solution containing:
- 0.1% (vol/vol) triethylamine (Millipore, TX1200)
- 0.2% (vol/vol) allyltrichlorosilane (Sigma, 107778)
- dissolved in chloroform
Wash 1x with chloroform
Wash 1x with ethanol
Bake in a 70 °C oven for 1 h to dehydrate the silane layer.
Silanized coverslips may be stored at RT in a desiccated chamber for weeks.

Coat silanized coverslips with 0.1 mg/mL poly-D-lysine (PDL) diluted in nuclease-free water for 1 min at RT.
UV-sterilized coverslips before plating cells.
Plate cells at desired density, allow to recover 1-36 hrs in incubator before fixation.
Fix with 4% (vol/vol) paraformaldehyde (PFA; Electron Microscopy Sciences, 15714) in 1× PBS for 20 min.
Wash three times with 1× PBS,
Permeabilize using 0.5% (vol/vol) Triton X-100 (Sigma, T8787) in 1× PBS for 10 min.
Wash three times with 1× PBS.
incubate for 5 min in a 30% formamide wash buffer, containing:
- 2× saline-sodium citrate (SSC; ThermoFisher, AM9763) and
- 30% (vol/vol) formamide (ThermoFisher, AM9342)
Hybridize encoding probes in encoding hybridization buffer, containing final conc. of:
- 2× SSC,
- 30% (vol/vol) formamide,
- 0.1% (wt/vol) yeast tRNA (Life Technologies, 15401-011) [Not sure this helps]
- 1% (vol/vol) murine RNase inhibitor (New England Biolabs, M0314L) [should not be necessary but often is]
- 10% (wt/vol) dextran sulfate (Sigma, D8906),
- in a humidity-controlled 37 °C incubator for 36 h.
- Recommended starting conc. of encoding probes: 100 μM for ~100 gene library.

prepare de-gassed PA-gel solution. Final conc:
- 4% (v/v) 19:1 acrylamide/bis-acrylamide
- 60 mM Tris-HCl PH 8
- 0.3 M NaCl
- fiducial beads (1:500 - 1:200,000)
- de-gas in vacuum chamber
Wash samples in PA-gel solution + polymerizing agents:
- ammonium persulfate. Final conc: (0.03% wt/vol)
- TEMED (0.15% v/v). Fincal conc: (0.15%)
add 50 uL of gel solution to a glass plate pretreated for 5 min with 1 mL of GelSlick
aspirated to remove excess PA gel solution
gently inverted onto this 50-μL droplet to form a thin layer of PA between the coverslip and the glass plate.
- (The volume of this gel droplet could be used to control the thickness of this PA film.)
allowed to cast for 1.5 h at room temperature.
coverslip and the glass plate were then gently separated, and the PA film washed twice with a digestion buffer consisting of:
- 0.8 M guanidine-HCl (Sigma, G3272),
- 50 mM Tris·HCl pH 8,
- 1mM EDTA, and
- 0.5% (vol/vol) Triton X-100
- in nuclease-free water
Digest proteins by covering with digestion buffer supplemented with 1% (vol/vol) proteinase K (New England Biolabs, P8107S) for >12 h in a humidified, 37 °C incubator.
wash with 2× SSC three times
MERFISH measurements should be performed immediately to minimize opportunity for RNA degradation.
- sample may be stored in 2× SSC supplemented with 0.1% (vol/vol) murine RNase inhibitor at 4 °C for no longer than 24 h.