[Histonet] In situ detection problems

[Jan Roger]

I am having serious problems with my RNA:RNA in situ hybridisation.

A couple of weeks ago, my result changed from having wonderful blue staining with NBT/BCIP, no background, to getting dirty brown slides with black precipitate, and much less staining. I have tried everything to avoid this problem including changing all buffers, baking glassware again etc., but no success (I have not tried changing antibody/ colour reagents, but different probe makes no difference). 

What am I missing? It cannot be a problem with my protocol in general, as it was working before. Is it a problem with tissue, detection reagents, antibody or probe? Any suggestions much appreciated.

A brief run-down of my protocol (buffer washes not included)
4 % paraformaldehyde (PFA)-fixed paraffin-embedded sections, rehydrated through alcohols.
20 ug/ml Proteinase K to remove cross-links
Post-fixation 5 min in 4 % PFA
10 min in 0.5 % acetic anhydride to acetylate sections

Hyb buffer:
4 x SSC
40 % deionized formamide
1x Denhardts
250 ug/ml ssDNA
2 mM EDTA
37oC overnight

Post-hyb washes:
2xSSC (2x15 min)
1xSSC (2x15 min)
0.1xSSC (2x30 min)
47oC

Antibody: 1 % Roche blocking reagent + 2% normal serum +1:200 anti-DIG-AP

Detection:
0.1 M Tris-HCl
0.1 M NaCl
0.05 M MgCl2
BCIP/NBT at manufacturers recommended dilution.
Overnight 4oC


Jan Roger
Division of Animal Physiology
Department of Biological Sciences
University of Nottingham
Sutton Bonington Campus
Loughborough
Leicestershire
LE12 5RD
Tel: 0115 9518862
Fax: 0115 9516302


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