11 Formalin-Fixed and Paraffin-Embedded Tissue Sections
151
- Xylol 100%
- Ethanol 100%, 85% and 70%, store at 4°C
- 1 M NaSCN, autoclaved
- 0.2 M HCI
- Pepsin, lyophilized 2500-3500 units/mg
- 0.9% NaCI, pH 2.0
- 2xSSC, pH 7.0 (prepare from 20xSSC)
- 70% Formamide in 2x SSC: Deionise formamide using mixed bed resins (e.g. Dowex, mix 70 ml deionised formamide with 10 ml 20 x SSC,
add distilled water to 100 ml volume, adjust to pH 7.0
- Formalin 3.7% in phosphate buffered saline
Third day: post-hybridization procedure
- 2xSSC/0.3% Nonidet p40
- DAPI staining solution: 0.2 f.lg DAPl/ml 2xSSC, prepare from stock
solution (200 f.lg DAPIIml distilled water)
- Mounting medium for fluorescence microscopy (e.g. Vectashield)
Procedure
First day: preparing specimen slides
1. Cut 5 f.lm thick paraffin sections using a microtome.
2. Float the section on a protein free water bath at 40°C.
3. Mount the section on slides.
4. Air dry the slides.
5. Bake the slides overnight at 56°C.
Reagents
and solutions
Reagents
and solutions
151
- Xylol 100%
- Ethanol 100%, 85% and 70%, store at 4°C
- 1 M NaSCN, autoclaved
- 0.2 M HCI
- Pepsin, lyophilized 2500-3500 units/mg
- 0.9% NaCI, pH 2.0
- 2xSSC, pH 7.0 (prepare from 20xSSC)
- 70% Formamide in 2x SSC: Deionise formamide using mixed bed resins (e.g. Dowex, mix 70 ml deionised formamide with 10 ml 20 x SSC,
add distilled water to 100 ml volume, adjust to pH 7.0
- Formalin 3.7% in phosphate buffered saline
Third day: post-hybridization procedure
- 2xSSC/0.3% Nonidet p40
- DAPI staining solution: 0.2 f.lg DAPl/ml 2xSSC, prepare from stock
solution (200 f.lg DAPIIml distilled water)
- Mounting medium for fluorescence microscopy (e.g. Vectashield)
Procedure
First day: preparing specimen slides
1. Cut 5 f.lm thick paraffin sections using a microtome.
2. Float the section on a protein free water bath at 40°C.
3. Mount the section on slides.
4. Air dry the slides.
5. Bake the slides overnight at 56°C.
Reagents
and solutions
Reagents
and solutions