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Electron Microscopy Sciences

Technical Data Sheets

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 sections
HRP 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
74,13g of sodium acetate
5,5 ml of glacial acetic acid
ad 500 ml Aqua dest.
0,1M SODIUM ACETATE BUFFER (pH 5,6)
50 ml of 2M sodium acetate stock solution
ad 1000 ml Aqua dest. (adjust pH to 5,6)
1M PHOSPHATE STOCK SOLUTION
112,5g Na2HPO4
30g KH2PO4
ad 1000 ml Aqua dest.
0,1M PHOSPHATE BUFFER (pH 6,5)
100 ml of 1M phosphate stock solution
ad 1000 ml Aqua dest. (adjust pH to 6,5)
0,01M PHOSPHATE PUFFER (pH 7,4)
10 ml of 1M phosphate stock solution
ad 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
100g of sucrose
10 ml of 10% NaN3 solution
ad 1000 ml Aqua dest. (pH 7,4)
1M TRIS STOCK SOLUTION
121,14g of tris
ad 1000 ml Aqua dest.
0,1M TRIS BUFFER (pH 9,4)
100 ml of 1M tris stock solution
ad 1000 ml Aqua dest. (adjust pH to 9,4)

Fixation solutions

Buffered 4% FORMALIN SOLUTION
100 ml of 37% formol
4g of NaH2PO4 H2O
6,5g of Na2HPO4
ad 1000 ml Aqua dest. (pH 7,0)
8% PARAFORMALDEHYDE STOCK SOLUTION
40g of paraformaldehyde
ad 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
1g light green yellowish
2 ml glacial acetic acid
ad 1000 ml Aqua dest.
PHOSPHOMOLYBDIC ACID / ORANGE G
30 g Phosphomolybdic acid
ad 500 ml Aqua dest.sodium nitrate in aqua dest.; vortex for 1 min; let react for 5 min.)
20 g Orange-G
ad 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)
ad 100 ml Aqua dest.
– boil
1 ml of glacial acetic acid
– Filtrate
   
Sol. B: 2 g of Ponceau de Xylidine
ad 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

Online Ordering

Technovit® 9100 Methyl Methacrylate is available online from the EMSCatalog. For ordering or product information, .