In situ hyb with Digoxigenin

Liz Jones E.Jones at sheffield.ac.uk
Fri Feb 9 12:38:41 EST 1996


K. Sayani wrote :
"I have previously conducted in situ hybridization using riboprobes
labeled via in vitro transcription with Digoxigenin-UTP as the label.
However, upon switching to a new probe, both sense and antisense probes
give me positive staining. I have played around unsuccessfully
with the stringency of the washes.  I either get the staining with both
probes or it all disappears." 

Are you detecting mRNA?  I had the same problem looking for cytokine 
mRNA in lymphoid tissue and paraffin embedded cell clots using 
dig-labelled oligonucleotide probe cocktails.    Many people i have 
spoken to have found the negative sense control to be positive (!)  so 
it is essential to include an RNAase digestion slide to ensure that this 
binding is RNA-specific. This will tell you if the positivity you are 
observing with the negative control probe is due to specific binding to 
an as yet unspecified RNA sequence or because of non-specific binding to 
protein/DNA/lipid.  To ensure that the RNAase digestion works, use a 
probe and positive control section which you have already shown to work, 
as background staining does not appear to be such a problem if an 
established probe which binds preferentially to its correct target 
sequence is used for ISH.  If the signal you are getting is non-specific 
then it may be that the probe cannot access its target if the tissue has 
been overfixed (?) or underdigested with proteinase K/pronase so 
optimisation of these parameters is CRITICAL.  I could not optimise 
tissue fixation so i found that ISH often gave inconsistent results 
between different experiments.  Are you sure that the RNA within your 
sections is intact?  Otherwise, it may be (as i found with my probes) 
that the probes for whatever reason do not bind the correct target 
perhaps because of secondary structure within the mRNA template? I wrote 
a chapter in my thesis on the problems you are experiencing although i 
never tried riboprobes, so feel free to contact me by e-mail if you need 
any more info/references.  Good luck!  Liz





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