Technovit® 9100 Short Instructions
EMS Catalog #14655
In-situ hybridization for sections
1. Prepare approx. 5µm-thick sections |
2. Section deplastization at room temperature• Xylol• 2-methoxyethyl acetate (2-MEA)• High-purity acetone• Aqua dest.If necesary deplasticize for more time, also without 2.MEA |
2 x 20 min 1 x 20 min 2 x 5 min 2 x 5 min |
3. Edge sections |
4. Block the endogenous peroxidase 3% H2O2 in methanol |
30 min |
5. Aqua dest. |
2 x 5 min |
6. Enzymatic digestion• Fast enzyme• Pronase 0,1%, 37°C |
10 min bei RT 10 min |
7. Aqua dest. |
minimum 10 min |
8. Fresh aqua dest. |
possibly also overnight |
9. Let sections dry |
10. Apply probe to the sections, cover with cover glass and seal with Fixogum |
11. Place sections in the hybridizer and hybridize for 2 hours at 55°C |
12. Remove sections from device, remove Fixogum |
13. Place sections in wash buffer |
2 x 2 min at RT |
Detection:
1. Place AP anti-biotin on the sections |
30 min, 37°C |
2. Wash in buffer |
2 x 2 min, RT |
3. Place AP substrate on the sections |
30 min, 37°C |
4. Wash in buffer |
2 x 2 min, RT |
5. Place HRP anti dig. on the sections |
30 min, 37°C |
6. Wash in buffer |
2 x 2 min, RT |
7. Place HRP substrate on the sectionsHRP substrate from kit for formalin and plastic-fixated iliac crests:HRP substrate for Shäfer fixated iliac crests:• 10,5 mg of 3-Aminoethylcarbazole (Sigma a 5754)• 1 ml DMSO• in 50 ml acetate buffer pH 5,6 (0,1 molar)• 5µl H2O2 |
30 min, 37°C |
8. Rinse sections under running water and if necessary counterstain briefly with diluted hemalaun. |
9. Cover with water |
NOTE:
Ask the corresponding probe manufacturer for additional instructions on ISH.
Reagents
Buffer
- 2M SODIUM ACETATE STOCK SOLUTION
- 74.13 g of sodium acetate
- 5.5 ml of glacial acetic acid
- add 500 ml Aqua dest.
- 0.1M SODIUM ACETATE BUFFER (pH 5.6)
- 50 ml of 2M sodium acetate stock solution
- add 1000 ml Aqua dest. (adjust pH to 5.6)
- 1M PHOSPHATE STOCK SOLUTION
- 112.5 g Na2HPO4
- 30 g KH2PO4
- add 1000 ml Aqua dest.
- 0.1M PHOSPHATE BUFFER (pH 6.5)
- 100 ml of 1M phosphate stock solution
- add 1000 ml Aqua dest. (adjust pH to 6.5)
- 0.01M PHOSPHATE PUFFER (pH 7.4)
- 10 ml of 1M phosphate stock solution
- add 1000 ml Aqua dest. (adjust pH to 7.4)
- 0.04M PHOSPHATE BUFFER + 10% SUCROSE (pH 7.4)
- 40 ml of 1M phosphate stock solution
- 100 g of sucrose
- 10 ml of 10% NaN3 solution
- add 1000 ml Aqua dest. (pH 7.4)
- 1M TRIS STOCK SOLUTION
- 121.14g of tris
- add 1000 ml Aqua dest.
- 0.1M TRIS BUFFER (pH 9.4)
- 100 ml of 1M tris stock solution
- add 1000 ml Aqua dest. (adjust pH to 9.4)
Fixation solutions
- Buffered 4% FORMALIN SOLUTION
- 100 ml of 37% formol
- 4 g of NaH2PO4 H2O
- 6.5 g of Na2HPO4
- add 1000 ml Aqua dest. (pH 7.0)
- 8% PARAFORMALDEHYDE STOCK SOLUTION
- 40 g of paraformaldehyde
- add 500 ml Aqua dest.
- 1.4% PARAFORMALDEHYDE SOLUTION
- 35 ml of 8% paraformaldehyde stock solution
- 65 ml of Aqua dest.
- 100 ml of 0.04M phosphate buffer +10% sucrose (pH 7.4)
Reaction batches
- FAST RED SOLUTION
- Put 3 ml of substrate buffer in a plastic tube
- Put 1 Fast Red tablet in the solution and dissolve
- Add 120 µl of levamisol and mix
- Sol. has a shelf life of 1 hour
- REACTION SOLUTION: ALKALINE PHOSPHATASE
- 50 ml 0,1M tris buffer (pH 9.4)
- 50 mg of Real Blue salt
- 25 mg of naphthol-AS-BI phosphate (dissolved in 0.5 ml of DMSO/Triton X 100)
- REACTION SOLUTION: ACID PHOSPHATASE
- 50 ml of 0.1M sodium acetate buffer (pH 5.6)
- 500 µl of hexonium pararosaniline (250 μl 4% pararsonanilin in 2N HCl + 250 µl of 4% sodium nitrate in aqua dest.; vortex for 1 min. let react for 5 min.)
- 25 mg of naphthol-AS-BI-phosphate (dissolved in 0.5 ml of DMSO/Triton X 100)
- REACTION SOLUTION: ASD-CHLOROACETATE ESTERASE
- 50 ml of 0.1M phosphate buffer (pH 6.5)
- 15 mg of naphthol AS-D chloroacetate (dissolved in DMSO/TritonX 100)
- 250 µl of hexonium pararosaniline
Staining solutions
- GIEMSA SOLUTION
- 3% sol., make using the stock solution (Merck)
- 1-2 drops of 1% acetic acid
- LIGHT GREEN
- 1 g light green yellowish
- 2 ml glacial acetic acid
- add 1000 ml Aqua dest.
- PHOSPHOMOLYBDIC ACID / ORANGE G
- 30 g Phosphomolybdic acid
- add 500 ml Aqua dest.sodium nitrate in aqua dest.; vortex for 1 min; let react for 5 min.)
- 20 g Orange-G
- add 500 ml Aqua dest.
- – Mix both solutions
– Filtrate
- PONCEAU ACID MAGENTA AZOPHLOXIN
- 100 ml Masson sol.
- 20 ml Azophloxinlsg.
- 880 ml 0.2% acetic acid
- Masson sol.: 1 part sol. A + 2 parts sol. B
-
Sol. A: |
1 g of acid magenta (magenta-S) add 100 ml Aqua dest. – boil 1 ml of glacial acetic acid – Filtrate |
|
|
Sol. B: |
2 g of Ponceau de Xylidine add 200 ml Aqua dest. – boil 2 ml of glacial acetic acid – Filtrate |
- AZOPHLOXIN SOLUTION
- 0.5 g azophloxin
- ad 100 ml Aqua dest.
- 2 ml glacial acetic acid
Source of Information
Self-experiment with reagents completed by Zytomed Systems GmbH
Product Information
Technovit® 9100 Methyl Methacrylate