Single molecule RNA fluorescent in situ hybridization (FISH) and imaging

All steps are done in RNase free conditions and with RNase free solutions and vessels.

Fixation of C. elegans
a) Add 5mL M9 buffer to a large plate (90mm) of age-synchronized worms. Gently rock plate to release worms from surface, then transfer worms to a 15mL conical centrifuge tube.
b) Spin down worms (4000rpm for 1 minute) and remove supernatant.
c) Add 5mL M9 buffer.
d) Spin down worms (4000rpm for 1 minute) and remove supernatant.
e) Add 1mL fixation buffer, transfer to microcentrifuge tube, and incubate at room temperature (RT) for 45 minutes.
f) Spin down worms (4000rpm for 1 minute) and remove supernatant, then add 1mL of 1X PBS.
g) Repeat step above to ultimately wash worms twice in 1X PBS.
h) To permeabilize, add 1mL of 70% ethanol and store overnight at 4 degrees Celsius with continuous rotation. Samples can be stored at 4 degrees in 70% ethanol up to a week before hybridization.
 

Hybridization of C. elegans
Steps should be performed in fume hood.
a) Centrifuge samples (4000rpm) and remove 70% ethanol.
b) Resuspend worms in 1mL of Wash Buffer A (made fresh) and incubate at RT for 2-5 minutes.
c) Centrifuge samples (4000rpm) and remove Wash Buffer A.
d) Add 100uL of the Hybridization Buffer containing probe (made fresh). Incubate overnight at 30 degrees Celsius in the dark.

Next day
a) Add 1mL of Wash Buffer A to sample. Vortex, spin down (4000rpm) and aspirate.
e) Resuspend in another 1mL of Wash Buffer A and incubate at 30 degrees Celsius, in the dark for 30 minutes.
f) Spin down (4000rpm) and aspirate Wash Buffer A, then add 1mL of Wash Buffer B. Incubate at RT for 2-5 minutes.
g) Spin down (4000rpm) and aspirate Wash Buffer B, then resuspend in a small amount (enough to cover the sample) of Wash Buffer B. Image immediately following hybridization.
 

Mounting worms
a) Pipette 2-5uL of the sample onto a clean 8mm round cover glass (Electron Microscopy Sciences, #1.5 thickness).
b) Gently tap a clean 22 x 22 mm square cover glass onto the drop of sample solution. This will cause the round cover glass to quickly adhere to the square glass.
c) Immediately flip the square glass so that the round glass is on top. Let sit for half a minute or so while covered in the dark.
d) While waiting, adhere a square Silicone Isolator (Grace Biolabs, 20 mm diameter x 0.5 depth) to a standard microscope slide.
e) Gently remove excess solution from the rim of the round cover glass with a Kimwipe.
f) Adhere the square cover glass to the Silicone Isolator with the round cover glass facing towards the microscope slide. Press down on the edges of the square glass to create a tight teal. Use tape to anchor sides of square coverslip down to microscope slide. This constitutes the imaging chamber.
g) Affix the imaging chamber to the microscopy stage. Make sure to position the microscope slide correctly so that the imagingchamber faces the objective.

M9 solution: (500mL)
Potassium Phosphate Monobasic (FW 136.09)         1.5g
Sodium Phosphate Dibasic Anhydrous (FW 141.96) 3g
Sodium Chloride (FW 58.44)                                      2.5g
1M Magnesium Sulfate (FW 120.37)                          500μl
Add Millipore treated water to bring up to 500mls
Autoclave solution

Fixation Buffer: (10mL)
Final composition is 3.7% (vol./vol.) formaldehyde in 1X PBS
For a final volume of 10mL, mix:
         1mL 37% Formaldehyde solution
         1mL 10X Phosphate Buffered Saline (PBS), RNase-free
         8mL Nuclease-free water
 

Wash Buffer A (10mL):
Final composition is 10% (vol./vol.) formamide in 1X Wash Buffer A
Mix and dilute Wash Buffer A fresh for each experiment.

For a final volume of 10mL, mix:
                  2mL of Stellaris RNA FISH Wash Buffer A (Biosearch Technologies Cat# SMF-WA1-60)
                  Add 7mL Nuclease-free water
                  Add 1mL deionized formamide
                  Mix well by vortexing gently

Hybridization Buffer: 1mL
Final composition is 10% (vol./vol.) formamide in Hybridization Buffer
Hybridization Buffer should be mixed fresh for each experiment.
             Due to the viscosity of the solution, recommend accounting for a 10% final volume excess in order to have enough Hybridization Buffer for all of your samples.

For a final volume of 1 mL, mix:
         900uL Stellaris RNA FISH Hybridization Buffer (Biosearch Technologies Cat# SMF-HB1-10)
         100uL Deionized Formamide

         NOTE: Do not freeze hybridization buffer
WARNING: Formamide is a teratogen that is easily absorbed through the skin and should be used in a chemical fume hood.
WARNING: Be sure to let the formamide warm to RT before opening the bottle.