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Kansas State University

DETECTION OF IN SITU HYBRIDIZATION (ISH) SIGNALS WITH STREPTAVIDIN

Suggested chemicals: DETEK–HRP Kit, Enzo Diagnostics Cat. No. 43820.

 

  1. Remove cover slips by dipping the slides in a 2 X SSC solution and letting the cover slips fall from the slides.
  2. Prepare the following hybridization mixture. 10 µL 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.
    3. 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
  3. 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).
  4. After the 1 X PBS wash, drain (but never dry) the slides on paper towels.
  5. Apply 100 µL 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:
    • 0.05% DAB (diaminobenzidine tetrahydrochloride) solution is 5 mg DAB in 10 mL 1 X PBS for 10 slides. Caution: DAB is a dangerous carcinogen.
    • 2 % Giemsa solution is 4-mL stock Giemsa solution in 200-mL H2O.
  6. Remove the cover slips by tilting or dipping the slides into a beaker containing 1 X PBS.
  7. Wash the slides in 1 X PBS at room temperature for 5 min.
  8. During the last 1 X PBS wash, add 4 µL H2O2 (hydrogen peroxide) to the 10-mL 0.05% DAB solution and mix well.
  9. 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.
  10. 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.
  11. 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 blue.