11.Wash slides in cold 2X SSC, followed by two changes of 2X SSC.
12.Dehydrate the slide through a cold alcohol series (70%, 90%, and 100% ethanol for 1 min each)..
13.Air dry the slides and place on a hot plate at ~42℃.
14.Apply 10mL of denatured probe mix to the slide.
15.Immediately apply a coverslip and seal with rubber cement. Keep the slide in a moist chamber at 37℃ overnight – four days for hybridization.
Washing slides
16. Place the wash tanks containing WS-1 in a 45℃ water bath for at least 30 minutes prior to use.
17. Remove rubber cement by using forceps. Coverslips can then be removed either by soaking in 2X SSC or gently tipping them off into the glass disposal bin (never pull them off!).
18. Wash the slides 3 times with WS-1 at 45℃ for 5 –10 min each (usu. 5min).
19. Wash the slides 3 times with WS-2 at RT for 2 min each.
20. (Optional) a) Blocking treatment with 1-3% BSA in 4X SSC for 20 min at RT.
21. b) Wash the slides 3 times with WS-2 at RT for 2 min each.
22. Wash the slides with WS-3 for 2 min at RT.
Note: make sure the slides are not dried in any point during the detection and washing steps.
Signal enhancement
23. Add 40uL Avidin-FITC (5ug/mL Avidin in PNM buffer) onto slides and cover with coverslip. Keep the slides in a moist chamber in dark for 20 min at RT.
24. Wash the slide as steps 19, 21 (do not perform step 18 & 20). Then, proceed from 24 to 26 steps or directly jump to steps 27-29.
25. (Optional): Add 40uL anti-Avidin (5ug/mL anti-Avidin in PNM buffer) onto slides and cover with coverslip. Keep the slides in a moist chamber in dark for 20 min at RT.
26. Wash the slides as steps 23.
27. Repeat steps 22-23 for one additional Avidin-FITC treatment.
Visualizing the hybridization
28. Dehydrate the slides with 70%, 90%, and 100% ethanol for 1-2 min each and allow to air dry.
29. Apply 40uL of DAPI II counterstain and a coverslip to hybridization location.
30. Store in dark if not use, otherwise, examine the slide at once under fluorescence microscope.
