DETECTION OF IN SITU HYBRIDIZATION (ISH)
SIGNALS WITH STREPTAVIDIN
Suggested chemicals: DETEK–HRP Kit, Enzo
Diagnostics Cat. No. 43820.
- Remove cover slips by dipping the slides in a 2 X SSC solution and
letting the cover slips fall from the slides.
- Prepare the following hybridization mixture. 10 ÁL of the hybridization
mix is used for one slide and covered with an '18 x 18' cover slip.
Care should be taken that the sloution is well mixed, because the
50% dextran sulfate solution is very sticky.
|2 X SSC at room temperature for 5 min,
|2 X SSC at 37°C for 10 min,
|2 X SSC at room temperature for 5 min, and
|1 X PBS at room temperature for 5 mi
- Prepare the detection soultion (horseradish peroxidase-conjugated
streptavidin) during washing: mix 900 µL H2O with
100 µL 10 X buffer and then add 3-10 µL of the streptavidin
solution (for 10 slides).
- After the 1 X PBS wash, drain (but never dry) the slides on paper
- Apply 100 ÁL of the detection solution on each slide and cover with
a '22 x 22' mm cover slip; then place the slides in the same moist
chamber used for the hybridization step and incubate at 37°C for
30 min. During this step, prepare the 0.05 % DAB and 2% Giemsa solution:
Remove the cover slips by tilting or dipping the slides into a beaker
containing 1 X PBS.
Wash the slides in 1 X PBS at room temperature for 5 min.
During the last 1 X PBS wash, add 4 ÁL H2O2
(hydrogen peroxide) to the 10-mL 0.05% DAB solution and mix well.
After the last 1 X PBS wash, drain the slides on paper towels and
apply 1 mL of the 0.05% DAP/H2O2 to each slide.
Do not cover with a cover slip. Incubate the slides
in the dark at room temperature for 10 min.
Remove the 0.05% DAP/H2O2 solution by tilting
the slides to paper towels and briefly rinse the slides in 1 X PBS.
Stain the slides in 2% Giemsa solution for 2 min.
Rinse the slides with tap water and dry with a hand puff (the slides
may be rinsed again if the Giemsa staining is too dark). Hybridization
signals appear light to dark brown in color, whereas chromosomes stain
- 0.05% DAB (diaminobenzidine tetrahydrochloride) solution is 5
mg DAB in 10 mL 1 X PBS for 10 slides. Caution: DAB is a dangerous
- 2 % Giemsa solution is 4-mL stock Giemsa solution in 200-mL H2O.