Daniel:Notebook/RNAFISH/2014-10-14

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ABP Trial Run II[edit]

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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]

  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, ~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)
  1. Prehybridize slides ~ 1 hour+ at 70C with prehybridization solution
  2. Denature probe at 90C for 10-15 minutes.
  3. Drop temp to 65C
  4. Add about 50-100 ng of probe to each slide in appropriate buffer; should be ~25 uL
  5. Seal coverslip with rubber cement, wait ~5 minutes for cement to dry
  6. 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