Daniel:Notebook/RNAFISH/2014-10-14
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ABP Trial Run II[edit]
Well, it would appear I made a mistake. The tissue specimen was apparently on the other side and I mistakenly did the protocol to the wrong section, so I need to redo the ABP protocol. One benefit did come from this. When I examined the correct side of the slide, with there still appeared to be a decent amount of signal. I may want to use something like NaBH4 to reduce autofluorescence.
Tissue Fixation[edit]
- Fix slides/sections in freshly prepared 4% paraformaldehyde/PBS depc, pH 7.2-7.4 at room temp. for 30 minutes
- Wash 2x in 2xSSPE for 5 minutes each
- Permeabilize in 0.6% Triton X-100/2xSSPEdepc for 30 minutes, room temp
- Wash 3 x 5 minutes in 2xSSPE
- Treat slides for 10 minutes/room temp in acetylation buffer
- Make the buffer just before use
- Wash 3x 5 mins 2xSSPE
- 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, ~15 minutes
Hybridization[edit]
Sample Matrix
Sample | Buffer | uL RBFOX3-488 (ng) | uL FOXP2-594 (ng) |
A | Long Cai | 2 (110) | 2 (120) |
B | Arjun Raj | 3 (110) | 3 (120) |
- Prehybridize slides ~ 1 hour+ at 70C with prehybridization solution
- Denature probe at 90C for 10-15 minutes.
- Drop temp to 65C
- 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 at 37 C
Buffers[edit]
- Prehybridization solution:
- 50% deionized formamide
- 50% 2xSSC pH 7.0
- Add dextran sulfate to 1%
- Acetylation Buffer; Make the buffer just before use:
- H2O/depc* 29.5ml
- Triethanolamine 0.4ml
- HCl (conc.) 52ul (microliters)
- Mix well; add 75ul acetic anhydride, stir to mix until globules are gone. Use immediately.
- Hybridization solution A-Long Cai
- 2X SSC
- 20% Formamide
- 10% Dextran Sulfate
- Hybridization solution B-Arjun Raj
- 2X SSC
- 10% Formamide
- 10% Dextran Sulfate
- 0.02% RNAse-free BSA
- 50 μg E.coli tRNA