In Situ Hybridization
Radioactive probe
5.0 ul 5xtranscription buffer
2.0 ul 0.1M DTT
5.0 ul DEPC H2O
1.0 ul linearized DNA (about 1ug)
1.0 ul 10mM CTP
1.0 ul 10mM ATP
1.0 ul 10mM GTP
1.0 ul RNase Inhibitor
7.0 ul 35S-UTP(Amersham)
Vortex and spin down
1.0 ul RNA Polymerase
Slightly vortex and spin down
1. Incubate radioactive probe for 2-3 hr at 37°C.
2. Add 1ul DNaseI to each reaction. Incubate 15min at RT. Prep column during this
step
3. Purification of probe on BioRad Micro Bio-spin6 Chromatography column( cat#
732-6200). Follow directions from the kit when using the column. Invert the
column sharply several times to resuspend the settled gel and remove any bubbles.
Snap off the tip and place the column in a 2.0ml microcentrifuge tube. Remove
the top cap, if the column doesn’t begin to flow, push the cap back on the column
and remove it again to start the flow. Allow the excess packing buffer to drain by
gravity to the top of the gel bed. Discard the drained buffer, then spin at 1000xg
for 2min, discard the buffer.
4. Add 50ul H2O to labeling reaction (total 75ul, if you have more than one reaction
of the same probe you can combine together up to 75ul before applying to the
column) and apply to the column, then spin at 1000xg for 4 minutes. Collect
aliquots
5. Radioactive probe count: count 1ul of aliquots. Labelled probe can be stored at 20°C for 1-2 days or -80°C for up to a week. Hottest---ideal 2x106 cpm,
usable >1x106 cpm.
Pre-hybridization, Hybridization and Post-hybridization
Pre-hybridization:
1. Remove slides from -80 and place direactly into 4% buffered formaldehyde for 1
hr at RT
2. Wash slide 3times in 2xSSC, 5min for each wash on orbital shaker.
3. Place slides in 0.1M TEA( PH=8.0, Triethanolamine) with 0.25% vol/vol acetic
anhydride( dropwise 500ul acetic anhydride to 200ml 0.1M TEA, mix well before
use), 10min at RT on stirring plate.
4. Rinse twice with ddH2O, 2min for each wash at orbital shaker.
5. Dehydrate sections in graded EtoH (50%, 75%, 95%, 95%, 100%,100%) and air
dry.
Hybridization
1. Prepare hybridization buffer per slide (50ul) contains:
(1) A=ul of Probe=Slides# X 1.2/ cpm
(2) B=Total Volumn=50 X Slide# X 1.2
(3) C=Amount of 100mM DTT=B X 0.1
(4) D=Amount of Cocktail=B-(A+C)
2. Put 50ul on a coverslip and pick up coverslip with slide to cover sections. Place in
a hybridization box with a 50% formamide soaked filter paper in the bottom (use
plastic rods to support slides so they are not touching filter paper). Wrap boxes in
saran wrap place in 55°C hybridization oven overnight.
Post Hybridization
1.
2.
3.
4.
5.
Remove coverslip by soaking in 2xSSC.
Wash 3x in 2xSSC (5 min each)
RNaseA incubation---better to use fresh stock, 200ug/ml at 37°C for 1 hr
Rinse in 2xSSC, 1xSSC at RT, 0.5xSSC for 5 min each.
High stringency wash 65°C for 1 hr in 0.1xSSC. (2ml 2xSSC+38ml dH2O>0.1xSSC)
6. Cool to RT.
7. Transfer to ddH2O to equilibrate for 5min at RT
8. Dehydrate sections in graded EtOH (50%, 75%, 95%, 95%, 100%, 100%).
Solutions for InSitu
1. 0.1M PB ( 200ml for 0.5M PB)
11.5 g Na2HPO4
2.54 g NaH2PO4
In 900ml H2O, adjust PH=7.4, then add H2O up to 1000ml.
2. 4% Formaldehyde
(1) Dissolve 2.76g NaH2PO4 (Monobasic) and 11.36g Na2HPO4 (Diabasic) in
400ml H2O
(2) Heat 500ml H2O to 60°C(No higher than 60°C), add 40g paraformaldehyde,
stir and drop 3 pellets NaOH until solution clears.
(3) Mix (1) and (2), stir until solution cools.
(4) Filter solution.
(5) Adjust PH=7.4, add H2O up to 1L and stored at 4°C
3. 1X TBS (PH=7.5)
Add 12.1g Tris Base and 8.77 NaCl in 900ml H2O, adjust PH=7.5 ( try to use
12N HCl to adjust, if 1N HCl, you will need a big volumn for that). Add H2O up
to 1L
4. 1X ASB (PH=9.5 without MgCl2) ( 100ml for 10X ASB)
Add 12.1 g Tris base and 8.77g NaCl in 900ml H2O, adjust PH=9.5 (use 1N HCl)
Add H2O up to 1L.
5. 1M Triethanolamine(TEA , PH=8.0)
Take 53.2 ml TEA in 300ml H2O, adjustPH=8.0 (Original PH=10.53, use almost
10ml 12N HCl to adjust), add H2O up to 400ml.
6. RNaseA Buffer ( PH=8.0)
1.38g Tris-HCl (10mM Tris-HCl )
29.0g NaCl (0.5M NaCl)
In 900ml H2O ( PH~5.6 ), adjust PH=8.0 (use 1N NaOH), then add H2O up to 1L.
7. RNaseA Stock (200ug/ml)
Add 10mg RNaseA in 50ml RNaseA Buffer (PH=8.0)