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HARALD RIEDER et al.
Second day: hybridization procedure
- Prewarm Coplin jar with NaSCN to 80°C in a water bath
- Prewarm a bottle with 0.9% NaCI and an empty Coplin jar in a 37°C
water bath
- Adjust the heating plate to approx. 47°C
It is advisable to place the Coplin jars onto a gently moving shaker during
incubation to improve the tissue-solution contact.
Deparaffinization 1. Immerse slides in Xylol: 3x 10 min.
2. Dehydrate slides in 100% ethanol: 2x 5 min.
3. Dry slides on a heating plate: 2-3 min.
Acid treatment Treatment with acid (e.g. HCI) pulls the basic histone proteins away from
DNA; especially in formalin fixed material, it helps to deproteinize the
DNA if signals are weak.
4. Immerse slides in 0.2 N HCI at room temperature: up to 20 min.
5. Rinse in distilled water: 3 min.
6. Rinse in 2x SSC: 3 min.
Chaotrope
treatment
Chaotrope treatment by a solvent disrupting molecular structures - which
is a compound or group of secondary structures that interfere with the
mechanism which stabilizes the complex proteins, nucleic acids and polysaccharides. These agents denature macromolecules primarily through
their disruption of the water lattice.
7. Incubate slides in 1 M NaSCN, 80°C: 30 min.
8. Rinse in distilled water: 1 min.
9. Immerse slides in 2xSSC at room temperature (solutions can be kept
for step B): 2xS min.
10. Turn down water bath to 73°C and place Coplin jar containing formamide into it.
Note: Caution: glass jars may crack if not prewarmed!
HARALD RIEDER et al.
Second day: hybridization procedure
- Prewarm Coplin jar with NaSCN to 80°C in a water bath
- Prewarm a bottle with 0.9% NaCI and an empty Coplin jar in a 37°C
water bath
- Adjust the heating plate to approx. 47°C
It is advisable to place the Coplin jars onto a gently moving shaker during
incubation to improve the tissue-solution contact.
Deparaffinization 1. Immerse slides in Xylol: 3x 10 min.
2. Dehydrate slides in 100% ethanol: 2x 5 min.
3. Dry slides on a heating plate: 2-3 min.
Acid treatment Treatment with acid (e.g. HCI) pulls the basic histone proteins away from
DNA; especially in formalin fixed material, it helps to deproteinize the
DNA if signals are weak.
4. Immerse slides in 0.2 N HCI at room temperature: up to 20 min.
5. Rinse in distilled water: 3 min.
6. Rinse in 2x SSC: 3 min.
Chaotrope
treatment
Chaotrope treatment by a solvent disrupting molecular structures - which
is a compound or group of secondary structures that interfere with the
mechanism which stabilizes the complex proteins, nucleic acids and polysaccharides. These agents denature macromolecules primarily through
their disruption of the water lattice.
7. Incubate slides in 1 M NaSCN, 80°C: 30 min.
8. Rinse in distilled water: 1 min.
9. Immerse slides in 2xSSC at room temperature (solutions can be kept
for step B): 2xS min.
10. Turn down water bath to 73°C and place Coplin jar containing formamide into it.
Note: Caution: glass jars may crack if not prewarmed!