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

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.