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
do preparations fall off slides? Why wash slides in acid before use?
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