5. Circular glass coverslips (#1.5, 12 mm): Treat with 2 M HCl
for 5 min, wash under a continuous stream of deionized water
for at least 15 min, immerse in 75% ethanol for 15 min, dry in a
biosafety cabinet to maintain sterility, and place it under UV for
1 h. Finally, store the dried coverslips in a sterile 10 mm petri
dish at room temperature. Seal with parafilm to prevent contamination (see Note 4).
6. 12-Well cell culture plates.
2.2 Digitonin
Extraction
1. 5% (w/v) Digitonin extraction buffer: Dissolve powdered digitonin in RNase-free Milli-Q water and store small aliquots at
À20
C until needed.
2. 0.025% Digitonin extraction buffer: Dilute 5% digitonin
extraction buffer 1:200 with warm CHO buffer immediately
prior to use.
3. CHO buffer: 115 mM KAc, 25 mM HEPES pH 7.4, 2.5 mM
MgCl 2 , 2 mM EGTA, and 150 mM sucrose in RNase-free
water. Store at 4
C (see Note 5).
4. Heat block set to 40
C: To ensure that the heat block is sterile
and RNase free, apply a few drops of deionized water to the
surface of the heat block and cover this with a layer of parafilm.
Remove any bubbles using RNase-free gloves by pressing
firmly onto the parafilm to ensure a flat working surface.
5. RNase-free filter tips.
6. PBS: 0.14 M NaCl, 0.003 M KCl, 0.01 M Na 2 HPO 4 , 0.002 M
KH 2 PO 4 , pH 7.4. Combine deionized H 2 O to a final volume
of 1 L.
7. 4% Paraformaldehyde fixing solution: Dilute 37% paraformaldehyde in PBS immediately prior to extraction.
8. 250 mL Beakers.
9. Methanol: Pour about 200 mL of methanol into a 250 mL
beaker and store at À20
C for at least 30 min prior to experiment (see Note 6).
10. 35 mm Cell culture dishes.
11. Kimwipes.
12. RNase-free gloves.
2.3 smFISH Staining
1. 10Â Saline sodium citrate (SCC) buffer: 1.5 M NaCl, 150 mM
sodium citrate, pH 8.0.
2. FISH hybridization buffer: 2Â SSC, 100 mg/mL dextran
sulfate, 1 mg/mL yeast tRNA, 5 mM vanadyl riboside complex, 10% formaldehyde. Make up to 1 L, and store aliquots of
1 mL at À20
C (see Note 7).
40
Jingze J. Wu and Alexander F. Palazzo
for 5 min, wash under a continuous stream of deionized water
for at least 15 min, immerse in 75% ethanol for 15 min, dry in a
biosafety cabinet to maintain sterility, and place it under UV for
1 h. Finally, store the dried coverslips in a sterile 10 mm petri
dish at room temperature. Seal with parafilm to prevent contamination (see Note 4).
6. 12-Well cell culture plates.
2.2 Digitonin
Extraction
1. 5% (w/v) Digitonin extraction buffer: Dissolve powdered digitonin in RNase-free Milli-Q water and store small aliquots at
À20
C until needed.
2. 0.025% Digitonin extraction buffer: Dilute 5% digitonin
extraction buffer 1:200 with warm CHO buffer immediately
prior to use.
3. CHO buffer: 115 mM KAc, 25 mM HEPES pH 7.4, 2.5 mM
MgCl 2 , 2 mM EGTA, and 150 mM sucrose in RNase-free
water. Store at 4
C (see Note 5).
4. Heat block set to 40
C: To ensure that the heat block is sterile
and RNase free, apply a few drops of deionized water to the
surface of the heat block and cover this with a layer of parafilm.
Remove any bubbles using RNase-free gloves by pressing
firmly onto the parafilm to ensure a flat working surface.
5. RNase-free filter tips.
6. PBS: 0.14 M NaCl, 0.003 M KCl, 0.01 M Na 2 HPO 4 , 0.002 M
KH 2 PO 4 , pH 7.4. Combine deionized H 2 O to a final volume
of 1 L.
7. 4% Paraformaldehyde fixing solution: Dilute 37% paraformaldehyde in PBS immediately prior to extraction.
8. 250 mL Beakers.
9. Methanol: Pour about 200 mL of methanol into a 250 mL
beaker and store at À20
C for at least 30 min prior to experiment (see Note 6).
10. 35 mm Cell culture dishes.
11. Kimwipes.
12. RNase-free gloves.
2.3 smFISH Staining
1. 10Â Saline sodium citrate (SCC) buffer: 1.5 M NaCl, 150 mM
sodium citrate, pH 8.0.
2. FISH hybridization buffer: 2Â SSC, 100 mg/mL dextran
sulfate, 1 mg/mL yeast tRNA, 5 mM vanadyl riboside complex, 10% formaldehyde. Make up to 1 L, and store aliquots of
1 mL at À20
C (see Note 7).
40
Jingze J. Wu and Alexander F. Palazzo