Daniel:Notebook/RNAFISH/2015-2-5: Difference between revisions
Jump to navigation
Jump to search
>Djacobse |
>Djacobse |
||
Line 9: | Line 9: | ||
#Allow cells to adhere for 6 hours at 37 C | #Allow cells to adhere for 6 hours at 37 C | ||
#Rinse slides briefly in 1X PBS | #Rinse slides briefly in 1X PBS | ||
#Fix slides for 5 | #Fix slides for 5 minutes in 4% (v/v) paraformaldehyde in 1X PBS | ||
#Wash 2x in 2xSSPE for 5 minutes each | #Wash 2x in 2xSSPE for 5 minutes each | ||
Revision as of 21:45, 5 February 2015
Cell Line RNA FISH
Slide Preparation
- Prepare a cell suspension of 500 000-2 000 000 cells/mL in culture media
- Add 20 uL of cell suspension per dish to the cener; add 80 uL culture media
- Allow cells to adhere for 6 hours at 37 C
- Rinse slides briefly in 1X PBS
- Fix slides for 5 minutes in 4% (v/v) paraformaldehyde in 1X PBS
- Wash 2x in 2xSSPE for 5 minutes each
Hybridization
- Permeabilize by leaving at room temperature for 30 minutes in 0.6% Triton X-100/2xSSPEdepc
- Wash 2x for 5 minutes in 2X SSPE
- Dehydrate in 30, 50, 70, 100, 100 ethanols 2 mins ea.
- Make solutions from absolute ethanol and depc/H20*.
- Dry in oven at 50-65C.
- Prehybridize slides ~ 1 hour+ at 70C with prehybridization solution
- Determine probe concentration (usually 2-4ng/ul) and denature probe at 90C for 10-15 minutes.
- Drop temp to 65C
- Spin briefly. Do not open the tube when it is at 90C-probe loss can occur.
- Add about 50-100 ng of probe to each slide in appropriate buffer; should be ~25 uL
- Seal coverslip with rubber cement, wait ~5 minutes for cement to dry
- Hybridize overnight appropriate 37C
- Prehybridization solution:
- 50% deionized formamide
- 50% 2xSSC pH 7.0
- Add dextran sulfate to 1%
- Hybridization solution (Arjun Raj)
- 2X SSC
- 10% Formamide
- 10% Dextran Sulfate
- 0.02% RNAse-free BSA
- 50 μg E.coli tRNA