Frequently Asked Questions
about in situ hybridization

Methods for DNA and RNA in situ hybridization are described in the book Practical In Situ Hybridization by Trude Schwarzacher and Pat Heslop-Harrison published by BIOS (Oxford)


  • Why do preparations fall off slides? Why wash slides in acid before use?
  • How quickly do fluorescent microscope lenses detriorate?


Why do preparations fall off slides? Why wash slides in acid before use?
Slide 'Washing': We clean new  microscope slides by soaking in chromic acid for 3 hours (to one month - two batches are always soaking). For years, we called this washing, but new slides cannot be seriously greasy/dirty. But we knew that omitting the step often lead to selective loss of metaphase spreads and poor in situ results. Now we think the soaking neutralizes the sodium hydroxide used in glass manufacture: without neutralizing, oil in the preparations will turn to soap, and the pH will change during hybridization (SSC is a poor buffer). The result also explains why some slide makes/batches do not need soaking - they have less residual NaOH.

How quickly do fluorescent microscopy lenses detriorate?
It seems that the high powers of UV used in fluorescent microscopy makes objective lenses autofluorescent. The worst cause of degredation is probably leaving the shutter open (with lens transmitting UV) when the microscope is not in use: we considered using foot switches to hold the shutter open. With our objectives, 5 yr old ones give an exposure time of 3 min with only epifluorescence on and no specimen, and have a clear yellow glow with UV light, while new ones give no reading. I know one company doing human clinical diagnosis that returns their objectives every 11 months for replacement under guarantee - which apparently happens without question. We suspect the glue holding the lens together is at fault and not the glass.