In Situ Hybridizations of Seeds

Mario Nenno [Biologie] nenno at rhrk.uni-kl.de
Thu Apr 22 10:56:55 EST 1993


Melissa Melan (mmelan at pattie.wellesley.edu) writes:

>Dear Networkers,
>        Does anyone out there have experience with in situ hybridizations in
>seed tissues?  (preferably Arabidopsis seed).  We are probing hydrated seed and
>early germination stage seed.  We have modified the fixation and infiltration
>steps of the paraffin sectioning to allow for slower movement of substances
>into and out of the seed tissues.  For the most part, stained sections before
>the hybridization steps look good (good cell morphology and good preservation
>of nuclei and cytoplasm).  However, AFTER hybridizations are completed it
>looks as if there are only cell walls left (i.e. very scant cytoplasm).  Has
>anyone else out there experienced similar problems?  Are we not fixing the
>tissue enough? (2% paraformaldehyde; 12-16 hrs)  Are we overdoing the ProteinaseK treatment? (1ug/ml; 37oC; 30 min)

Hallo Melissa,

perhaps you should shorten the incubation time when using proteinase K
in this concentration. Try 7 to 8 min to start with.
Check your slides after proteinase K incubation before proceeding 
with in situ hybridization.

Hope this helps,
      _____________  
     / Mario Nenno \\
 ___/               \\___________________________________________
| Universitaet Kaiserslautern       6750 Kaiserslautern, Germany |
| FB Biologie/Abt. Zellbiologie   Internet: nenno at rhrk.uni-kl.de |
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