Dig In Situ Trouble Shooting Guide

Dr. Bruce Link link at SHIVA.PSU.EDU
Thu Jun 17 13:37:40 EST 1993


Over the past five months I have been speciallizing a digoxigenin protocol for
arabidopsis vegetative meristems.  The technique which I use is based largely on
the protocol by Ann Hirsch ("In-Situ Hybridization of Nodulin mRNAs in Root
Nodules Using Non-Radioactive Probes", Birgit Bochenek and Ann M. Hirsch, Plant
Molecular Biology Report,Volume 8(4):237-248).  However I have been modifying
the procedure based on techniques I have read about in other protocols, or have
picked up from talking with people in other labs.

I am currently writting up a more formal protocol, since there seems to be
some interest in this.  For the time being here is a trouble shooting guide
for Digoxygenin in situs:




			Trouble Shooting Guide for Digoxigenin
			Labled RNA In Situ Hybridizations.

Apperance of Sense Slides:

	For Best Resuls:

	Sense Slides should be very clean with no expression anywhere.
	Sometimes faint expression appears.  If it does it should be diffuse,
	and definitely not dark. Xylem elements generally stain before
	other tissues.  If this happens possible sources are:

			-poor ab blocking
			-probe conc. too high
			-too much time  in ab step
			-ab conc. too high
			-color reaction too long

Dark sections around edges of slide:

	-sections dried out while changing solutions
		(absolutely avoid this!)


Anti-Sense Slides:
	
	Variable Expression Patterns:

		1. Air bubbles:

		    These are generally noticeable in that they affect some
		    sections and not adjacent sections.  Variable resuls 
		    across the whole slide could occur if you get lots
		    of bubbles under the coverslips after each change of 
		    solutions.

		2. Expression (or dark expression ) at the edges of the slide:

		    This is most often caused by drying. Any section that
		    is allowed to dry after starting into the prehyb step 
		    will stain darkly. Sometimes these patterns are quite
		    interesting, tissue specific, and ,of course, meaningless.

		3. Variable Expression across the whole slide.  Weak or 
		    inconsistent patterns:

			a) Poor tissue fixation:

				this generally resuls in high signal
				to noise ratios on the antisense slides
				giving mudding expression patterns, or
				worse no expression patterns at all.

				Tissues must be fixed in fresh 4% Formaldahyde
				(made from paraformaldehyde). Dehydration steps 
				should be done the following day, followed by
				embedding.  The tissues aren't safe until they
				are fully embedded.  Each day is a drop in
				signal intensity.

				We use the fixation and embedding technique of
				Enrico Coen ("Floricaula: A Homeotic Gene 
				Required for Flower Development in Antirrhinum
				majus",Cell 63:1311-1322) Developed by David
				Jackson ("In situ Hybridization in plants" in 
				Molecular Plant Pathology: A Practical Approach,
				D.J. Bowles, S.J. Gurr, and M. McPhereson, eds.
				England: Oxford University Press), in press
				1990

				Note: To visuallize the tissues in paraffin they
				may be stained with  0.1% Eosin Y in 95% Ethanol

			b) Poor Deparaffinization:

				Generally muddy appearing expression patterns
				with background on the slide where there are
				no sections.  The expression patterns can do
				anything here, and are probably determined 
				mostly by the local paraffin coating.

				Some tissues such as Cauliflower are
				particularly difficul to deparaffinize, and
				require extra, or longer xylene treatments.
				This is not a problem with Arabidopsis

			c) Dust:

				In the Hirsch protocol coverslips are changed
				five times.  They must be clean. Slides must
				be clean. Even dust in bottles used to mix up
				solutions can be a problem.  Dirt always sticks
				to the sections you are interested in.  It can
				either increase or decrease expression patterns.


			d) Uneven probe concentration:

				When the hyb mix is applied to the slide there
				will always be pockets which are lower in probe
				concentration.  This is do to incomplete mixing
				of the prehyb and hyb mix on the slide.  It
				is better to leave some prehyb mix on the slide
				rather than waste time getting it all off. 
				While you are getting the prehyb off the slide,
 				your sections are drying, so you have to leave
				some prehyb on the slides.  You can reduce the
				concentration effect two ways: 
				  1) apply the hyb mix containing the probe
				     all along the bottom edge of the slide.
				     Next lay the coverslips down width wise,
				     i.e. bring the coverslip into full contact
				     with the bottom edge of the slide, and 
				     gently lay it down the short way, from
				     the bottom to the top of the slide.  This
				     helps avoid air bubbles (so I use this
				     technique for all coverslip changes) and
				     pushes the concentration differences to 
				     the top of the slide.  Since I always
				     lay my sections down near the bottom of
				     the slide this moves the concentration
				     gradient away from the sections.

				   2) If possible use a high concentration of
				     probe (e.g. 0.5 ng/ul). This will reduce
				     the effect of a localized hyb dilution
				     (1/2 of infinity = infinity). I have used
				      high concentrations with no background
				      problems.

				    3) A third method of reducing the 
				       concentration effect is to raise and
				       lower the cover slip as you are laying
				       it down, but before it is completely
				       down.  This helps to mix the hyb
				       and prehyb. Note: if you have about
				       three cups of strong coffee right before
		        	       you change cover slips you will do this
				       naturally.
				




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