Ver. 6.0
in situ Hybridization Protocol in a 96-well plate
(I) Sectioning
Plate preparation
1) Add 30 μl of 2% Vectabond (VECTOR) in EtOH and incubate for 10 min at RT.
2) Drain and wash with DEPC water for 2 times.
3) Drain and let them air dry at 35°C.
Procedure
1) Place tissues in ice-cold 4% PFA-PBS and leave at 4°C overnight
2) Wash DW for 30 min at 4°C
2) Replace solution with 50% EtOH-DW, 75%, and finally 100% EtOH, each for 2 h at RT.
3) Incubate in 50% PoX-EtOH for 2 h at RT, and finally at 45°C for 30 min
4) Incubate in 100% PoX at 45°C overnight
5) Transfer to 96 well flexible plate containing PoX and orientate as required, allow to set at room temperature.
6) Store at -70°C.
7) Cut ribbon of 8 μm sections on microtome at 25°C.
8) Mount the sections on a well of 96-plate coated gelatin containing 50 μl DW.
9) Drain the DW by using multi-pipet
10) Allow to dry at 30°C for overnight
1. Embedding material
Polyester Wax (PoX) (BDH) 100 g
Cetyl Alcohol (Sigma) 50 g
(II) Probe labeling
Template preparation
1. Pfu polymerase 1μl /ml
2. Primers that can amplify DNA cloned in BlueScript KS
KS-7U: 5’-TGGAATTGTGAGCGGATAAC
KS-7L: 5’-CTGGCGAAAGGGGGATGTGC
x 1 Pfu Buffer
x 10 Pfu Buffer 40 ml
dNTPs (20mM) 4 ml
Primer 7U (10000p/μl) 20 μl
Primer 7L (10000p/μl) 20 μl
DW up to 400 ml
x10 pfu Buffer
100 mM KCl
100 mM (NH4)2SO4
200mM Trisi-HCl (pH8.75)
20 mM MgSO4
1% Triton X-100
1mg/ml BSA
5. Glassmilk 4G (BIO 101, 2078-205), 250 μg DNA/ 166mg glass / ml solution , Unifilter (Whatman, 7700-4301),
Glasmilk 4G (5μg /100 μl) 100 ml
DNA Binging Buffer 400 ml
DNA Binging Buffer
4 M Guanidin thiocyanate
1 M NaCl
0.1 M Tris-HCl pH 7.0
New wash sol.
20mM Tris-HCl (pH7.0) 1 M 5 ml
50mM NaCl 5 M 5 ml
1mM EDTA 0.5 M 1 ml
60% EtOH 300 ml
DW 189 ml
Tatal 500 ml
80% EtOH
100% EtOH
1) Amplification of cloned cDNAs by the PCR using KS-7U and -7L in a 50 μl reaction vol in 35 cycle reactions (94°C 30 sec, 55°C 30 sec, 72°C 2 min) from colony-picked up templates.
2) DNA purification by the glass powder method by the 96 well format.
1. Dispense 75 μl Glassmilk into each well.
2. Mix the 50 μl of the PCR fragment.
3. Aspirate. ゆっくり引くこと
4. New wash x2 times
5. 80% EtOH wash 1x times
6. 100% EtOH
7. Centrifuge at 3000 rpm for 5 min.
8. Evaporate.
9. Add 50 μl of 5 mM Tris-HCl pH.8.0, 0.01% TritonX-100 incubate for 5 min.
10. Centrifuge at 2500 rpm for 10 min.
Transcription reaction was carried out in a 96-well U-plate
Digoxigenin-11-uridin-5’-triphosphat (DIG-11-UTP) (Boehringer) (Cat. No. 1209256)
Antisense Probe: T7 RNA polymerase
Sense Probe: T3 RNA polymerase
Dig NTP Mix (2.5 mM each)
DIG-UTP (10mM) 100 μl 25.0 250 500
UTP (100mM) 18.6 μl 4.65 46.5 93
ATP (100mM) 28.6 μl 7.15 71.5 143
CTP (100mM) 28.6 μl 7.15 71.5 143
GTP (100mM) 28.6 μl 7.15 71.5 143
DW 939.6 234.9 2349.0 4698
Total 1144 μl 286 μl 2860 μl 5720
Dig NTP Mix (2.5 mM each) with biotin
DIG-UTP (10mM) 100 μl 25.0 250 500 625
UTP (100mM) 18.6 μl 4.65 46.5 93 116.25
ATP (100mM) 28.6 μl 7.15 71.5 143 178.75
CTP (100mM) 28.6 μl 7.15 71.5 143 178.75
GTP (100mM) 28.6 μl 7.15 71.5 143 178.75
DW 939.6 234.9 2349.0 4698 5872.5
biotin-UTP (10 mM) 6.25
Total 1144 μl 286 μl 2860 μl 5720
1 plate 4 plate 8 plate 16 plate
x 110 x 420 x 840 x 1680
(22) (84) (168) (168 x 2)
x 5 Buffer 0.80 88.0 336.0 672.0 1344.0
DIG NTP Mix 0.64 70.4 268.8 537.6 1075.2
RNase Inhibitor 0.08 8.8 33.6 67.2 134.4
DTT (0.1M) 0.15 16.5 63.0 126.0 252.0
Enzyme (50U/ml) 0.032 3.52 13.44 26.88 53.76
BSA (2mg/ml) 0.10 11.0 42.0 84.0 168.0
DNA 2.2 μl
Total 4.0 μl
Incubate in 37°C CO2 incubator for 2h
Stop the reaction by addition of 10 μl tRNA (4mg/ml), 50 μl of LiCl-EtOH (4M LiCl 7.5 ml, EtOH 225 ml) and incubate for 1 h at -20°C, centrifuge at 3,000rpm for 20 min, drain, and finally suspend in 100 μl RNA suspending buffer (dial 5).
RNA suspending buffer:
20 mM Tris-HCl 5 ml
20 mM DTT 1.45 g
0.5% SDS 12.5 ml of 20%
Total 500 ml
(III) Hybridization
Preparing sections.
1) Add 100 μl of 100% EtOH and drain, repeat once more, drain completely.
2) Incubate the plate for 30 min at 35°C.
3) Add 100 μl of 50% EtOH-DW, drain.
4) Add 100 μl of PBST and drain
5) Add 75 μl of Proteinase K (10 μg/μl, x 100) in PBST, incubate at 25°C for 10 min drain
6) Add 100 μl of active DEPC-PBS (1/1000) and incubate for 15 min, repeat once more.
7) Wash with 100 μl of PBST
8) Add 100 μl of hybridization buffer, mix at dial 5 for 10 sec and incubate at 55°C for 120 min (prehybridization).
Probe dilution and hybridization washing
1) Add 1 μl probe to each prehybridized well and mix (30 sec at dial 5).
3) Incubate at 55°C for 16 h.
4) Wash 3 times of 5 cycles with 300 μl of TBST (20 mM Tris-HCl, 500 mM NaCl, 01% Tween20) and add 100 μl of TBST.
5) Incubate for 1 h at 55°C.
5) Drain and add 50 μl of Hybri Wash Buffer (20 mM Tris-HCl, 50 mM NaCl, 0.1% Triton, and 50% formamide), drain and add 100 μl of Hybri Wash Buffer again and incubate at 55°C for 2 h.
5) Wash two times of 5 cycles with TBST (20 mM Tris-HCl, 500 mM NaCl, 0.1% Tween20) .
6) Block in 0.5% Casein-1% Normal sheep serum TBST for 30 mim at RT.
Hybridization Sol
50% Formamide 250 ml
5x SSC 125 ml
0.5 mg/ml Heparin 250 mg
200 μg/ml yeast total RNA 1 ml of 100 mg/ml
100 μg/m ss-DNA
Total 500 ml
total RNA (100mg/ml, Sigma) and Heparin (Sigma H3393)
III. Detection.
TBST
NaCl 292 g
2 M Tris-HCl pH 7.5 100 ml
10% Tween 20 100 ml
Total 10 L
0.5% Casein-NSS-TBST
Casein (Hammarsten, BDH, 440203) 2.5 g
DW 50 ml
1 N NaOH 10 ml
heat by electric oven
TBST up to 480 ml
2 M Tris-HCl 10 ml
1 N HCl 10 ml
0.05% azide (Sigma, T-8784) 5 ml of 5% azide
5% Normal sheep serum 25 ml
filtrate with 0.45 filter
Anti-digoxigenin-AP, Fab fragment (Boehringer, 1093274)
NBT/BCIP Stock Solution (Boehringer, 1681451) x 50 conc.
Buffer 3.
100 mM Tris-HCl pH 9.5
100 mM NaCl
50 mM MgCl2
100 mM Levamisole (x100 conc.) 240 mg/10 ml store at 4°C
NBT/BCIP Stock Solution 100 μl (1/50)
Buffer 3 5 ml
100 mM Levamisole 50 μl (1/100)
5% azide 25 μl (1/200)
Filtrate by 0.45 μm Filter
Acetone powder from the tissue that is the same of the section
Anti-digoxigenin-AP, Fab fragment is absorbed by the acetone powder.
Acetone powder 10 mg
Casein-TBS 1 ml
Anti-Dig-AP (x500) 10 μl incubate for 30 min at RT
Centrifuge at max and harvest the sup.
Dilute in 30 ml Casein-TBS and filtrate by 0.7 μm filter
Procedure
1) Drain the block sol. from the plate and add 50 μl of diluted antibody and incubate for 2 h at 37°C.
2) Wash three times with TBS-T (5 times each), each for 10 min.
3) Drain and add 50 μl NBT/BCIP Solution.
4) Incubate for 6-8 h at 37°C incubator and stop the reaction by replacing to TBS.
NBT/BCIP Solution
和光 NBT 144-01993 1g x1
和光 BCIP 028-08663 1g x1
NBTに17.7ml のDWを加える。BCIPに35.4mlのDMSOを加える。
NBT 17.7ml 、BCIP 17.7m, DMSO17.7mを混ぜ合わせる。遮光低温保存。
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