Daniel:Notebook/RNAFISH/2014-10-14: Difference between revisions
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##Make solutions from absolute ethanol and depc/H20*. | ##Make solutions from absolute ethanol and depc/H20*. | ||
#Dry in oven at 50-65C. | #Dry in oven at 50-65C. | ||
===Buffers=== | |||
*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. |
Revision as of 20:29, 14 October 2014
ABP Trial Run II
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
- 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.
Buffers
- 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.