3-dimensional in situ hybridization
Materials and solutions
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PBS 1X
1L: 900ml H20, 100ml PBS 10X
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PFA 4% in PBS 1X
Needs to be at RT
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4-well or 8-well chamber slides
Slide staining jars (Coplin type)
Slide rack
Horizontal glass jar (Schifferdecker type)
Liquid nitrogen in a benchtop container
For 100ml: Heat 80ml of water in a fume hood with stirring to approximately 65C, making sure the
water does not boil. Add 40µlof NaOH 10M (allows PFA to dissolve). Then add 8g of
paraformaldehyde.
When solution is clear, add 10ml of PBS 10X and adjust volume to 100ml with water. The 8%
solution will be diluted to 4% with PBS 1X.
Adjust quantity in order to make stocks. Aliquot everything in 50ml conical tubes, store at -20oC
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0.5% Triton X-100 in PBS 1X
100ml: 10ml PBS 10X
2.5ml Triton X-100 20%
87.5ml H20
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20% Glycerol in PBS 1X
261ml: 60ml Glycerol
26.1ml PBS 10X
174.9ml H20
This solution will go in the horizontal glass jar. Use for all the liquid nitrogen-glycerol
permeabilization cycles.
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HCl 0.1M
100ml: 850µl HCl (37% fuming solution)
100ml H20
Solution should be made fresh each time.
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SSC 2X
100ml: 10ml SSC 20X
90ml H20
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Formamide 50% in SSC 2X at pH7.0
100ml: 10ml SSC 20X
90ml H20
Adjust the solution to pH 7.0 using Citric acid 1M or HCl 1M.
Pour the solution in the ‘Storing’ Coplin jars and place at 4oC.
The solution needs to be cool to preserve the slides.
Protocol
Day 1
1.
Passage cells and harvest them
2. Adjust number of cells. For 4 chamber slide: myoblast-8k, fibroblast-20k.
3. Seed cells onto 4 chamber or 8 chamber slides.
4. Put everything back in the incubator for the cells to attach.
Day 2
Remove media and chamber separators from the slides.
1.
Put all slides in the slide rack.
For all steps and washes, when transferring the slide rack from one jar to another, remove
the excess of solutions by gently tapping on paper towels.
2. PBS 1X
5 min
There will be multiple PBS washes, when transferring the slide rack from one jar to
another, remove the excess of solutions by gently tapping on paper towels.
3. PFA 4% in PBS
10 min
4. PBS 1X
5min
5.
20min
0.5% TritonX-100 in PBS 1X
3 times
First round of permeabilization
6. 20% Glycerol in PBS 1X
7.
30min
Liquid Nitrogen/ 20% Glycerol in PBS 1X
6 times
Place slides in the liquid nitrogen.
Wear gloves if needed. Wait until the liquid nitrogen stops bubbling-one should hear a
crisp sound if the area is not noisy.
Remove the slides from liquid nitrogen. Wait until completely thawed.
Incubate in the glycerol solution for 1min.
8. PBS 1X
5min
9. HCl 0.1M
10min
Allows access to the nuclei
10. SSC 2X
5min
11. End point: Remove slides from the slide rack, tap off excess solution, and store them in the
formamide solution in the Coplin jar at 4C. Wait a minimum of 5 days before moving the
experiment forward.
Be very careful with the jars. Solution is greasy. Put a small piece of paper towel beneath
the jar, wrap once with parafilm, and a final wrap with aluminum foil. Tape everything.
Put date/name of cells/experiment and store.