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

Back to Calendar

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

  1. Fix slides/sections in freshly prepared 4% paraformaldehyde/PBS depc, pH 7.2-7.4 at room temp. for 30 minutes
  2. Wash 2x in 2xSSPE for 5 minutes each
  3. Permeabilize in 0.6% Triton X-100/2xSSPEdepc for 30 minutes, room temp
  4. Wash 3 x 5 minutes in 2xSSPE
  5. Treat slides for 10 minutes/room temp in acetylation buffer
    1. Make the buffer just before use
  6. Wash 3x 5 mins 2xSSPE
  7. Dehydrate in 30, 50, 70, 100, 100 ethanols 2 mins ea.
    1. Make solutions from absolute ethanol and depc/H20*.
  8. 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.