7 In Situ Hybridization for RNA: Radioactive DNA Probe
105
1. Phosphate Buffered Saline (PBS)
0.2 M Phosphate buffer, pH 7.4
NaCI
Dilute to 1000 ml
2. 2 X PBS
0.2M Phosphate buffer, pH 7.4
NaCI
Dilute to 1000 ml
3. 10% Paraformaldehyde
50 ml
8.77 g
10 ml
1,75 g
Dissolve 8 g of paraformaldehyde in 80 ml of distilled water,
heat the mixture to about 60°C, add 1 N NaOH drop-wise, with
occasional agitation, until the mixture becomes clear. Cool to
room temperature.
4. 4% Paraformaldehyde in PBS "Fixative"
Mix 80 ml of 10% paraformaldehyde (Solution 3 ) with 100 ml
of2 x PBS (Solution 2). Adjust pH to 7.4 and dilute to 200 ml.
Store at room temperature.
Subbed slides for in situ hybridization
1. Incubate microscope slides in the following solution overnight at 60°C.
20x SSC
50 x Denhardt's mixture
H20
Total
30 ml
4 ml
166 ml
200 ml
Final Cone.
3x SCC
Ix Denhardt
Solutions
required