Waldir, 

At 10:19 21.03.00 -0300, you wrote:
>    Hello.
>
>    I work with  insects gut epithelium  and   I have had the same
>problem with PI, ToTo and BoBo, that stain all  nuclei and cytoplasm.
>    Would it be possible for someone to tell me which concentration of
>RNAase I should use in order to avoid inespecific staining?
>    Thanks.
>
>
>Waldir Caldeira
>IBUSP
>SÃO PAULO/BRASIL
>

Protocol for Staining of fixed, permeabilized specimens with PI
According to the Laboratory Manual of Maniatis et al. (second ed., Vol 3:,
B17)

---------------------------------------------
Use DNAse-free RNAse A (!) Stock solution prepared as follows:
(According to the Laboratory Manual of Maniatis et al., second ed., Vol 3)

RNAse A (e.g from SIGMA): dissolve 10 mg/ml in 10 mM Tris-HCl pH 7.5, 15 mM
NaCl
heat 15 min to 100°C
cool down slowly (!! with some heat insulation wrapped around the reagent
tube, to enable re-folding of RNAse)
store aliquots at -20°C

----------------------------------
RNAse teatment and DNA staining:

******** sequential, rapid stain:

cells on cover slip: add 100 µl RNAse A in PBS (RNAseA: DNAse-free, 10 U
per ml)
incubate for 30 min at 37°C
wash briefly in PBS pH 7.4 (room temp.)
wash briefly in PBS pH 8.9
embed in medium (e.g in Citifluor or Vectashield) containing PI (5 ug/ml)
seal with nail hardener (colorless)

******** alternatively: simultaneous stain (overnight):
cells on cover slip: add 100 µl RNAse A (DNAse-free, 10 U per ml) / PI (5
ug/ml)
incubate overnight at 0-4°C
wash briefly in PBS pH 7.4
wash briefly in PBS pH 8.9
embed in medium (e.g in Citifluor or Vectashield) containing PI (5 ug/ml)
seal with nail hardener (colorless)
You may omit the PBS wash steps (replace by 1x PBS pH 8.9 and 5 ug/mlPI in
the embedding medium.

In my hands, overnight stain with 10 ug PI/ml, without subsequent washing,
gave good staining (quantitative, as checked by flow cytometry of
suspension cells). But consider: the higher the free PI concentration, the
more likely you may get diffuse (unbound PI) background, because unbound PI
also shows a (very low) fluorescence.


Guenter
----------------------------------
Dr. Guenter Giese
MPI fuer Medizinische Forschung
Abt. Biomedizinische Optik
Jahnstr. 29
D-69120 Heidelberg
Phone (Germany or 0-)6221-486-320
Fax   (Germany or 0-)6221-486-325
e-mail: [log in to unmask]
------------------------------------------