| 1. template preparation step |
a) for cDNA insert in transcription
vector including promoter for SP6, T7,
or T3 RNA polymerase
|
- linearize cDNA with restriction enzyme
- purified linearized cDNA by ethanol precipitation
|
| b) for PCR-generated fragments
|
- prepare primers, such as SP6, T7, or T3 promoter sequence
- generate PCR fragments using PCR reaction
- purify PCR fragments using a commercially available kit
|
| 2. probe construction step |
- prepare cRNA probe reaction mixture
template (1- 2 ug) 1 ul
appropriate RNA polymerase 2
10x transcription buffer 2
10x DIG RNA labeling mix 2
sterile RNase free water 13
total volume 20
- incubate reaction mixture for 2 hours at 37 degree
- add 2 ul DNase I
- incubate for 15 minutes at 37 degree
- add 2 ul 0.2 M EDTA
|
| 1. deparaffinization and hydration step |
a) for cDNA insert in transcription
vector including promoter for SP6, T7,
or T3 RNA polymerase
|
- dry slides for 1 hour in an oven at 60 degree
- dewax slides in xylene for 3 min x 10 times
- hydrate slides in 100% ethanol for 2 min x 5 times and
70% ethanol for 2 min x 5 times
- immerse slides in DEPC-treated PBS for 5 minutes
|
| 2. postfixation step |
- immerse slides into 4% buffered paraformaldehyde for
10 minutes
|
| 3. proteinase digestion step |
- immerse slides in 10 ug/ml proteinase K solution for 20
minutes at room temperature
|
| 4.
quencing step of endogenous alkaline phosphatase |
- immerse slides in 0.2 N HCl for 1 hour
- wash slides in DEPC-PBS for 5 minutes
|
| 5. electrostatic interaction inhibition step |
- Immerse slides in 0.1 M TEA for 10 minutes
- immerse slides in 0.1 M TEA including 0.25% acetic acid
for 10 minutes
- wash slides in DEPC-PBS for 5 min x 2 times
- dry slides on air
|
| 6. Hybridization step |
- prewarm hybrisol for 20 minutes at 50 degree prior to
mix probe
- mix DIG-labeled probe (2 ug/ml) with prewarmed hybrisol
- incubate slide with probe for 16 hours at 50 degree on
HYBrite instrument
|
| 7. washing step |
- immerse slides in 2X SSC/50% formamide for 15 minutes
at 50 degree water bath
- remove coverslip from slides
- wash slides in 2X SSC/50% formamide for 30 min x 2 times
at 50 degree water bath
- wash slides in 2X SSC for 20 minutes at room temperature
- wash slides in washing buffer (or DIG1 solution) for
5 minutes at room temperature
|
| 8. incubate slides with blocking solution (or
DIG2 solution) for 2 hours |
9. blot excess blocking solution from section,
and incubate with anti-DIG antibody for 2 hours at room
temperature or overnight at 4 degree
|
| 10. wash slides in washing buffer (or DIG1 solution)
including tween-20 for 30 min x 4 times on shaker |
| 11. detection step |
- incubate slides in NBT/BCIP solution with 2 M MgCl2 and
1 mM levamisole or 4.5 ul NBT and 3.5 ul BCIP solution in
1 ml DIG3 solution with 25 ul MgCl2 and 1 mM levamisole
- stop the reaction by washing in DIG4 solution for 10 minutes
|
| 12. If it is needed, counterstain in methylgreen
|
| 13. mount with glycerol gels |
