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NARC - UiO
In Situ hybridization
In Situ protocols
Yeast Two-hybrid analysis
Yeast Two-hybrid protocols
 
 
www.narc.no
NARC - UMB
NARC - NTNU
 
 

Norwegian Arabidopsis Research Center

Please contact:
Reidunn B. Aalen
or
Ellen Andersen Department of Molecular Biosciences
Pb 1041, N-0316 Oslo, Norway.
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IN SITU PROTOCOLS

PROBE SYNTHESIS

NB All solutions, tips and tubes must be RNase free.

Linearization of plasmid

  • Digest the plasmid to completion, using an excess of enzyme in 100 ul reaction volume, overnight incubation
  • Check the digestion on agarose gel.
  • Add 100 ul DEPC-dH2O and extract 1x with 200 ul RNase-free Phenol:Chloroform:Isoamylalcohol 25:24:1 and 1x with Chloroform:Isoamylalcohol
  • Add 1/10 vol 3M NaOAc and 2,5 vol cold 100% Etanol
  • Spinn 15 minutes at 4ºC
  • Wash the pellett with cold 70% Etanol, spinn 5 minutes at 4ºC
  • Resuspend in 10-15 ul DEPC-dH2O, the solution must be concentrated (0.5 ug/ul?)
  • Check concentration on agarose gel.

Transcription

Mix:
linearized plasmid (3-5 ul, ca 2 ug)
5 ul 5x transcription optimized buffer
2 ul 10x DIG Labeling mix
1 ul RNasin
1 ul polymerase - T7, T3 or Sp6
DEPC-dH2O to 23 ul (plasmid + DEPC-dH2O = 14 ul)
  • Incubate 45 minutes at 37ºC
  • Add 1 ul RNasin og 1 ul polymerase
  • Incubate 45 minutes at 37ºC
  • Check yield on agarose gel.

DNase-treatment

  • Add 75 ul DEPC-dH2O, 1 ul tRNA [100mg/ml] and 2 ul DNase I
  • Incubate 10 minutes at 37ºC
  • Add 100 ul 4M NH4OAc og 400 ul 100 % Etanol
  • Precipitate at -20 ºC, preferably overnight, or at -80 ºC for at least 1 hour
  • Spinn 20 minutes at 4ºC
  • Wash the pellett with cold 70% Etanol, spinn 5 minutes at 4ºC
  • Resuspend in 50 ul DEPC-dH2O
  • Check result by agarose electrophoresis of a small sample.

FIXATION OF TISSUE

Fixation solution:50% Etanol, 5% Acetic acid, 3.7% Formaldehyde
Harvest tissue and immediately place in ice cold freshly made fixation solution. Fix under 20-25 In Hg vacuum for 15 minutes, release vacuum slowly. Incubate at room temperature for 1,5 hrs.

DEHYDRATION, STAINING AND EMBEDDING

50% Etanol 30 minutes
60% Etanol 30 minutes
70% Etanol 30 minutes
80% Etanol 30 minutes
95% Etanol with 0.1% Eosin Y   over night
100% Etanol 30 minutes
75% Etanol/25% Histoclear   30 minutes
50% Etanol/50% Histoclear 30 minutes
25% Etanol/75% Histoclear 30 minutes
100% Histoclear 3 x 1 hour

Add about 20 Paraplast chips and incubate at 42ºC over night. Add more Paraplast and continue incubation until all is melted. Move vials to 55ºC and continue incubation. Melt a large supply of Paraplast and keep at 55ºC. Change the Paraplast every morning and evening, total of 6 changes. Pour into plastic weigh boats and let harden. Make sure to orient the tissue before the Paraplast hardens to ease orientation during sectioning.

SLIDE TREATMENT AND HYBRIDIZATION

Dewax Histoclear 2x10 minutes
Hydrate Etanol series
2x100%
95%
85%/0.85% NaCl
70%/0.85% NaCl
50%/0.85% NaCl
30%/0.85% NaCl
15%/0.85% NaCl
0.85% NaCl
DEPC-dH2O		 2 minutes each
Acidify 0,2 M HCl 20 minutes
Wash DEPC-dH2O0
PBS
DEPC-dH2O		 5 minutes each
Proteinase K 1ug/ml in TE 30 minutes at 37ºC
Wash PBS 2 minutes
Glycine Block 2 mg/ml in PBS 2 minutes
Wash PBS 1 minute
Refix tissue 4% Paraformaldehyde in PBS, pH 7,0 20 minutes
Dehydrate Etanol series 2xDEPC-dH2O
0.85% NaCl
15%/0.85% NaCl
30%/0.85% NaCl
50%/0.85% NaCl
70%/0.85% NaCl
85%/0.85% NaCl
95%
2x100%		 2 minutes each
Hybridization [6xSSC, 3%SDS, 50% Formamide, 100 ug/ml tRNA]
50-200 ng probe		 2 days at 55ºC
Quick wash [0,2xSSC, 0,1%SDS]
High Stringency Wash [0,2xSSC, 0,1%SDS] 2 x 10 minutes at 55ºC
Wash 2xSSC 2 minutes
RNase treatment 10 ug/ml in 2xSSC 30 minutes at 37ºC
Wash 2xSSC 2 minutes
High Stringency Wash [0,2xSSC, 0,1%SDS] 2 x 10 minutes at 55ºC
Wash 2xSSC 2 minutes
Rinse TBS 5 minutes
Blocking 0,5% Blocking agent (DIG-kit) in TBS 2 x 45 minutes
Rinse TBS 1 minute
Antibody a-DIG AP diluted 1:1000 in 0,5% BSA in TBS 2 hours
Rinse TBS 3 x 5 minutes
Detection preincubation [10 mM Tris pH 9.5, 10 mM NaCl, 50 mM MgCl2] 5 minutes
Detection Western Blue solution with 1 mM Levamisol 2-6 days in the dark

Inspect the slides to monitor the color reaction. When sufficient signal has developed, the color reaction can be stopped by dehydration series of Etanol and the slides mounted with Cytoseal or Permount.

Reference protocol











Last update 04.12.06 by Barbro E. Sæther