Dual In Situ
DIG probe
Radioactive probe
10.5ul H2O
5ul transcription buffer
2.5ul 0.1M DTT
1ul UTP mix(80% Dig-11-UTP Roche 1209256;
20% Cold UTP 10mM)
1ul 10mM ATP
1ul 10mM CTP
1ul 10mM GTP
1ul Linearrized DNA (about 1ug)
1ul RNase Inhibitor
1ul RNA Polymerase (Roche T7, T3, SP6)
4.0ul H2O
5 ul transcription buffer
2.5ul 0.1M DTT
7.5ul 35S-UTP(Amersham)
1ul 10mM CTP
1ul 10mM ATP
1ul 10mM GTP
1ul linearized DNA (about 1ug)
1ul RNase Inhibitor
1ul RNA Polymerase
1. Incubate DIG probe for 1.5 hr at 37°C, incubate radioactive probe for 1.5-2hr 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. Add 1ul of 1M DTT to the probe
solution. 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.
6. DIG probe blot: Dot 1ul of DIG probe on a piece of Nytran. UV crosslinking at
Energy900 or dry for several hours. Quick rinse in 0.1MPB (phosphate buffer,
PH=7.4) in small beaker. Block with immuno buffer ( Fresh prepare) for 1-4hrs
(use 2hrs) at RT. Dilute digoxigenin antibody (Fab fragments with alkaline
phosphatase, Roche 1093274) to 1/20000 in fresh immuno buffer ( take 0.5ul of
10:1 diluted antibody in 1 ml immuno buffer) and incubate blot overnight at RT
with shaking.
7. Wash blot 3 times with 0.1M PB (2min for each wash), then wash with TBS for
10 min and wash with ASB (with MgCl2, 10.2mg MgCl2/ml ASB) for 10min
8. Make 1ml color reaction buffer ( 1ml ASB without MgCl2 + 5 ul NBT/BCIP,
Roche 1 681 451)
Place blot in solution in dark (inside the drawer). Don’t agitate, react for 10
minutes. Rinse extensively in water and air dry. Choose the darkest DIG-probe for
hybridization.
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 3 times in 2XSSC, 5 min for each wash.
3. Place slides in 0.1M TEA( PH=8.0, Triethanolamine) with 0.25% vol/vol acetic
anhydride( dropwise 75ul acetic anhydride to 30ml 0.1M TEA, mix well before
use), 10min at RT on stirring plate.
4. Rinse twice with water, 2min for each wash
5. Dehydrate sections in graded EtoH (50%, 70%, 95%, 95%, 100%) and air dry.
Hybridization
1. Pooling the hottest DIG probe. Best to use probe same day making it. For dual in
situ, hybridize radioactive and DIG probe at same time.
2. Assuming 1ul DIG probe is approximate to 1X106 cpm of radioactive probe. Can
try different concentrations of DIG probe because it tends to be less sensitive.
1X=2-3ul per slide
2X=4-6ul per slide
3. Prepare hybridization buffer per slide (50ul) contains:
X ul DIG probe
Y ul radioactive probe
0.5ul 0.1M DTT(final conc. of 10mM)
Bring up to 50ul with 50% Formamide Cocktail Buffer.
4. 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 coverslips by soaking in 2xSSC.
Wash 3x in 2xSSC (5min each, sloshing a bit)
RNaseA incubation---better to use fresh stock, 200ug/ml at 37°C for 1 hr
Rinse in 2x, 1x, 0.5x at RT, 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 0.1M PB in Coplin jar to equilibrate for 10min
8. Blocking----Incubate in immuno buffer for at least 1hr at RT with shaking
9. Antibody---Incubate overnight at RT with gently shaking in Digoxigenin antibody
diluted to 1/20000 ( 1ul AB in 20ml immuno buffer). Put lids on.
Washing
1. Do washes in Coplin jar with a slide/slot to ensure thorough washing. Try to use
different jars for AB and color steps. If not, make sure wash well.
2. Wash in 0.1MPB---2x for 30min
1xTBS ------ 2x for 30min(can’t wash too much here)
1xASB ------ 1x for 20min (long enough to equilibrate)
(50ml 1xASB+510mg MgCl2)
Color Reaction
1. Need at least 20ml per coplin jar. Heat 18ml water to 90°C, add 1g
PVA(Polyvinyl alcohol, sigma P-1763—final concentration is 5%) slowly with
stirring until in solution. Cool to RT. Add 2ml 10X ASB(PH=9.5 without MgCl2)
2. For each 20ml, add 4.8mg levamizole (Sigma L-9756) and 300ul NBT/BCIP
(Add right before putting slides in. Be carefully these are carcinogenic, Roche 1
681 451). Transfer slides to color reaction. Incubate in dark (Cover coplins with
foil) with no agitation. Check the slides for signal---30 min for abundant probes,
1-2hrs or overnight for less abundant probes (1hr for us). Check the results under
the microscope.
3. When sections reach the desired intensity, wash extensively in water (15min) to
stop the reaction
4. Dehydrate sections in graded EtOH (50%, 70%, 95%, 95%, 100%).
5. Dip in Xylene and permount coverslip, air dry for at least 24hr.
Post Color Reaction (For Dual InSitu)
1. After washing extensively in water for 15 min to stop the reaction, strip off AB to
reduce background with 0.1M Glycine, PH=2.2, 0.5% TritonX-100 (in water)
10min at RT.
2. Fix in 2.5% Glutaraldehyde (in water) for 1-2hr to stabilize reaction.
3. Rinse well in water for 15 min, quick ethanol dehydration, air dry, lay on film.
Emulsion dip
Emulsion dip sections in Ilford K5D ( PolySciences, Inc. Cat#:17537) at 37°C in dark
room without safety light, air dry slides for a couple of hours, wrap the slides box
with foil and store at 4°C (Stored time is based on the signal strength, roughly equals
to 3X radioactive exposured time), then develop in D-19 (M-store, 650328) for 2min
at RT, wash for 20 seconds in H2O and fix (M-store, 650456) for 3min, wash
extensively with water and dehydrate in graded EtOH, dip in xylene, coverslip with
permount.
Solutions for Dual InSitu (Use Meijer’s H2O)
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. Immuno Buffer
70ml
0.5% TritonX-100
350ul
0.25% Carrageenan
175mg ( dissolved in 25ml H2O first )
0.1 M PB
14ml 0.5M PB
Store in fridge, make fresh every experiment. Dissolve carrageenan in water first
and spin solution at RT for several hrs. then add PB.
7. 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.
8. RNaseA Stock (200ug/ml)
Add 10mg RNaseA in 50ml RNaseA Buffer (PH=8.0)
9. 0.1M Glycine (PH=2.2) / 0.5%TritonX-100 (MW=75.07)
Weight 3.75g Glycine in 450ml H2O, adjust PH=2.2, add H2O up to 500ml.
10. 2.5% Glutaraldehyde
Take 750ul Glutaraldehyde in 30ml H2O
Note:
1.
Double in situ
POMC: Color Reaction (T7)
CRHR1: Radioactive Reaction(T3)
3. SRIF: Color Reaction (SP6)
CRHR1 : Radioactive Reaction(T3)
Regular in situ
CRHR1:Radioactive reaction (T3)
CRHR1 : Radioactive Reaction(T3)