35. 1Â Phosphate-buffered saline (PBS): 137 mM NaCl, 2.7 mM
KCl, 8 mM Na 2 HPO 4 , and 2 mM KH 2 PO 4 .
36. 4% PFA-PBS solution.
37. Immunofluorescence solution (IF solution): 0.1% RNase-free
bovine serum albumin (BSA) in PBS.
38. Primary antibody: Resuspend the alpha-tubulin monoclonal
antibody in 2 mM Na azide, 1% BSA, and PBS.
39. Secondary antibody: Goat anti-mouse IgG (H + L) crossadsorbed secondary antibody conjugated to Alexa Fluor
647 or other fluorophores.
40. Mounting solution with the blue fluorescent DNA staining
agent 4
0 ,6-diamidino-2-phenylindole (DAPI).
41. Transparent nail polish.
42. Wide-field epifluorescence microscope (see Note 2).
43. smFISH analysis software (see Note 3).
44. Imaging processing software (see Note 4).
3 Methods
3.1 Coverslip
Washing and Coating
1. Use a 1 L beaker to boil two packages (~200 pieces) of microscope coverslips in 500 mL of 0.1 N HCl for 20–30 min.
Gently stir to separate the covers.
2. Rinse the coverslips ten times with water, autoclave, and keep
them at 4
C in 70% ethanol for up to a year.
3. On the day of the smFISH-IF, place the coverslips on a clean
chromatography paper, air-dry the ethanol and rinse with
water, aspirate the excess water, and air-dry. Treat the coverslips
for 20 min at room temperature (RT) with 200 μL of 0.01%
(w/v) poly-L-lysine. Aspirate the poly-L-lysine solution and let
the covers air-dry. Wash three times with DDW and allow to
air-dry.
4. Use forceps to place each coverslip, with the poly-L-lysinecoated side up, into a single well of a 6-well culture dish and
store the dish at RT. Coverslips need to be completely dried.
3.2 Growth, Fixation,
and Permeabilization
of Yeast Cells
1. At the appropriate temperature (25–30
C), grow overnight a
low-density yeast culture in SC complete and then dilute it
again in 25 ml of fresh medium to an OD 600 ~0.1; allow to
grow until OD 600 0.3–0.4. At this point, fix the cells (see
Note 5).
56
Evelina Tutucci and Robert H. Singer
KCl, 8 mM Na 2 HPO 4 , and 2 mM KH 2 PO 4 .
36. 4% PFA-PBS solution.
37. Immunofluorescence solution (IF solution): 0.1% RNase-free
bovine serum albumin (BSA) in PBS.
38. Primary antibody: Resuspend the alpha-tubulin monoclonal
antibody in 2 mM Na azide, 1% BSA, and PBS.
39. Secondary antibody: Goat anti-mouse IgG (H + L) crossadsorbed secondary antibody conjugated to Alexa Fluor
647 or other fluorophores.
40. Mounting solution with the blue fluorescent DNA staining
agent 4
0 ,6-diamidino-2-phenylindole (DAPI).
41. Transparent nail polish.
42. Wide-field epifluorescence microscope (see Note 2).
43. smFISH analysis software (see Note 3).
44. Imaging processing software (see Note 4).
3 Methods
3.1 Coverslip
Washing and Coating
1. Use a 1 L beaker to boil two packages (~200 pieces) of microscope coverslips in 500 mL of 0.1 N HCl for 20–30 min.
Gently stir to separate the covers.
2. Rinse the coverslips ten times with water, autoclave, and keep
them at 4
C in 70% ethanol for up to a year.
3. On the day of the smFISH-IF, place the coverslips on a clean
chromatography paper, air-dry the ethanol and rinse with
water, aspirate the excess water, and air-dry. Treat the coverslips
for 20 min at room temperature (RT) with 200 μL of 0.01%
(w/v) poly-L-lysine. Aspirate the poly-L-lysine solution and let
the covers air-dry. Wash three times with DDW and allow to
air-dry.
4. Use forceps to place each coverslip, with the poly-L-lysinecoated side up, into a single well of a 6-well culture dish and
store the dish at RT. Coverslips need to be completely dried.
3.2 Growth, Fixation,
and Permeabilization
of Yeast Cells
1. At the appropriate temperature (25–30
C), grow overnight a
low-density yeast culture in SC complete and then dilute it
again in 25 ml of fresh medium to an OD 600 ~0.1; allow to
grow until OD 600 0.3–0.4. At this point, fix the cells (see
Note 5).
56
Evelina Tutucci and Robert H. Singer