i am trying to find the best method of fixation for plant cell culture
material in in situ hybridization experiments with Dig-labeled riboprobes.
A fixation in 3-4% FA pH9 leads to nice signal after detection with
Mouse-Anti-Dig and Anti-Mouse-Fitc but to poor preservation of cell structure.
After Fixation in 3-4% FA and 0.1% GA the preservation is nice, but due to the
autofluorescence of GA-fixed cells i have to treat the cells with borohydride
(5mg/ml) for quenching. This treatment seems to eat away not only the
background but also the signal. Is this due to the reaction of borohydride
with all double bonds (nothing left to hybridize against) ? Who has a nicer
fixation protokoll, so that the preservation survives all the high temperature
inkubations of in situ experiments?
Any suggestions welcome!
Thank you in advance.