Fluorescence In Situ Hybridization of Paraffin-Embedded Formalin-Fixed Tissue Sections

user user at user.com
Mon Jun 7 15:04:28 EST 2004


We have been doing FISH with 300 bp Y chromosome probes in which we make 
the probe up with 11-digoxigenin-dUTP (using the Vysis kit) and then 
illuminate using direct fluorophoric (rhodamine-conjugated) antibody to 
Dig.

Now we have had no problem having consistent and continual success doing 
with cultured cell positive controls from the males, and cultured cell 
negative controls with the females (other negative controls include 
incubating cells with no Y probe---only competitive DNA).

But when we get to paraffin-embedded formalin-fixed tissues, there has 
not even been one success, let alone repeated success.

I have cardiac tissue from males and females for testing the 
effectiveness

I have tried many variables.  The method I am using now for the 6-micron 
heart sections is (all steps at ambient temp unless specified):

1. Clearing of sections:  Histoclear (xylene substitute), 3 successive 
    jars
     this is invariable since the paraffin must be removed
2. 100% absolute ethanol
     to wash away xylene
3. 0.2 N HCl for 20 minutes
    this is rather common apparently, but can it depurinate DNA and 
    affect hybrid formation/stability?
4. 1.0 M thiocyanate at 80 degrees
    heat treatment supposed to break some methylene bridges that formed 
    by formaldehyde fixation, make tissues more accessible
    have done heat treatment in pH 6.0 citrate in past (also well known 
    alternative)
5. protease digestion
    have tried:
   * pepsin at 0.5 mg/ml in 10 mM HCl, 37 deg for 30 min
   * proteinase K at 5 ug/ml in 20 mM Tris pH 7.4, 2 mM CaCl2 for 15 min
   * proteinase K at 15 ug/ml in Tris/CaCl2 for 30 min

The rest of the steps are rather uncontroversial.  Denaturation is 
soaking slide 3 min at 73 deg in 70% formamide/2X SSC then drying in 
ethanol series and placing on 45-50 degree slide warmer.  The probe is 
heated at same temp for 5 min in 50% formamide/2XSCC/10% dextran sulfate, 
and then spotted on dried target, left overnight in 37 deg humified 
chamber for hybridization (coverslips sealed by rubber cement prevent 
drying out).  Posthyb washing is 50% formamide/2XSSC, then 2XSSC, then 
2XSSC/0.1% NP-40, each two jars in succession, all at 46 degrees.  The 
anti-Dig antibody binding phase begins by blocking with PBS/0.1% Tween-
20/1% BSA, then with the antibody in PBS/Tween-20/0.2% BSA, and then 
washing with PBS/Tween-20.  The DAPI/antifade is put on, and then slide 
visualized.

The nuclei show up well with DAPI.  There appears to be some serious 
background red fluorescence in the tissue as we look with the TRITC 
filters.  The characteristic red dot in the nuclei does not show up 
consistently though.  We don't expect to get 100% of the nuclei in male 
heart sections to have red-dotted blue nuclei, and just blue nuclei in 
the females.  But published reports suggest that more than half the cells 
in the male sections should be unambiguously positive (100% of our male 
cultured cells are positive, but they go through a different fixation 
process:  after swelling nuclei with 75 mM KCl and fixing in 3:1 
MeOH:acetic acid, these are dropped onto slides and steamed a little 
before air-drying).

Other considerations??

1. Clearing:  we don't think that clearing is a problem.  Should sections 
be rehydrated gradually in a reverse ethanol series?

2. Withhold the mineral acid treatment?  Probably not a significant 
factor.

3. Tissue section thickness:  perhaps 6 microns is TOO thin, and one will 
see DAPI-stained nuclei but the part of the nuclei that was Y-bearing has 
been shaved off.  That would give misleading results.  But certainly we 
would expect many of the other cells to bear Y-chromosomes and be 
positive.

4. Heat/chemical treatments to improve probe accessibility.  This may be 
the major factor.  Protease and acid treatments have been done that seem 
to affect nuclear structure (DAPI staining with hollowed-out centers).

5. Indirect antibody illumination?  Using indirect antibody detection 
often increases background, but increases likelihood of detection.  But 
if conditions are excellent for cultured cells, why should they not be 
the same for tissue sections?

If anyone else has been confronted with this problem, I'd appreciate some 
suggestions.



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