8. Use forceps to place the coverslips, facing up, back into a
12-well plate containing 2 mL of pre-warmed pre-hybridization solution. Cover the 12-well plate with aluminum foil.
Incubate for 15 min at 37
C.
9. Aspirate the solution carefully, replace with 2 mL of
pre-warmed pre-hybridization buffer, and incubate again for
15 min at 37
C.
10. Wash once with 2 mL 2Â SSC/0.1% Triton X-100 solution for
5 min at RT (see Note 12).
11. Wash once with 2 mL of 2Â SSC for 5 min at RT.
12. Wash once with 2 mL of 1Â SSC for 5 min at RT.
3.4 Immunofluorescence (IF)
1. Fix the coverslips with 2 mL of 4% PFA in PBS for 10 min
at RT.
2. Wash once with 2 mL of PBS for 5 min at RT.
3. Prepare an IF chamber using a 150 mm petri dish (Fig. 2b).
Glue to the bottom of the dish the inverted cap of 1.5 mL
tubes. Use the caps to place the coverslips face up.
4. Incubate the coverslips in 190 μL of IF solution for 30 min at
RT (prepare 200 μL IF solution per coverslip).
5. Fill a 50 mL beaker with PBS till the top.
a
b
smFISH chamber
upside
coverslip
DDW
IF chamber
upsidedown
coverslip
DDW
Fig. 2 Example of smFISH or IF chambers. (a) A humidified chamber for smFISH is created by wrapping a
15 cm petri dish with aluminum foil and by covering the bottom of the dish with parafilm. A 15 mL plastic cap
filled with DDW is placed toward the edge of the dish. The dish is closed and sealed with parafilm to make a
hybridization chamber and to avoid evaporation of the hybridization mix. (b) A humidified chamber for IF is
prepared by covering a 10 cm petri dish with aluminum foil. The coverslips are placed upside on an inverted
1.5 mL tube cap glued to the bottom of the dish to act as a stand. DDW is added at the bottom of the dish
(~5 mL). The dish is closed and incubated at RT
Detection of mRNA and Proteins in Single Cells by smFISH-IF
59
12-well plate containing 2 mL of pre-warmed pre-hybridization solution. Cover the 12-well plate with aluminum foil.
Incubate for 15 min at 37
C.
9. Aspirate the solution carefully, replace with 2 mL of
pre-warmed pre-hybridization buffer, and incubate again for
15 min at 37
C.
10. Wash once with 2 mL 2Â SSC/0.1% Triton X-100 solution for
5 min at RT (see Note 12).
11. Wash once with 2 mL of 2Â SSC for 5 min at RT.
12. Wash once with 2 mL of 1Â SSC for 5 min at RT.
3.4 Immunofluorescence (IF)
1. Fix the coverslips with 2 mL of 4% PFA in PBS for 10 min
at RT.
2. Wash once with 2 mL of PBS for 5 min at RT.
3. Prepare an IF chamber using a 150 mm petri dish (Fig. 2b).
Glue to the bottom of the dish the inverted cap of 1.5 mL
tubes. Use the caps to place the coverslips face up.
4. Incubate the coverslips in 190 μL of IF solution for 30 min at
RT (prepare 200 μL IF solution per coverslip).
5. Fill a 50 mL beaker with PBS till the top.
a
b
smFISH chamber
upside
coverslip
DDW
IF chamber
upsidedown
coverslip
DDW
Fig. 2 Example of smFISH or IF chambers. (a) A humidified chamber for smFISH is created by wrapping a
15 cm petri dish with aluminum foil and by covering the bottom of the dish with parafilm. A 15 mL plastic cap
filled with DDW is placed toward the edge of the dish. The dish is closed and sealed with parafilm to make a
hybridization chamber and to avoid evaporation of the hybridization mix. (b) A humidified chamber for IF is
prepared by covering a 10 cm petri dish with aluminum foil. The coverslips are placed upside on an inverted
1.5 mL tube cap glued to the bottom of the dish to act as a stand. DDW is added at the bottom of the dish
(~5 mL). The dish is closed and incubated at RT
Detection of mRNA and Proteins in Single Cells by smFISH-IF
59