***In situ Preparation of Nuclear Matrix for Microscopy and
Immunoblotting***

*Equipment and reagents*

· Tissue culture dishes (35 mm and 150 mm)

· Tissue culture plates (6-well)

· Glass coverslips (22X22 mm, No: 1.5) stored in 75% (v/v) ethanol

· Phosphate-buffered saline (PBS)

· 5X SDS sample buffer

· 2% paraformaldehyde in PBS, prepare fresh and keep on ice

· Cytoskeleton (CSK) buffer \[100 mM NaCl, 300 mM sucrose, 10 mM Pipes,
pH 6.8, 3mM MgCl~2~,~\ ~store in aliquots at --20°C. Before use add the
following; 0.5% Triton X-100, 1 mM PMSF or AEBSF, Leupeptin 10 M,
Pepstatin A 10 M, E-64 15 M, Bestatin 50 M, Recombinant RNasin (Promega)
ribonuclease inhibitor 20 units/ml\], after reconstitution keep on ice.

· Extraction buffer \[250 mM ammonium sulfate, 300 mM sucrose, 10 mM
Pipes, pH 6.8, 3 mM MgCl~2~,~\ ~0.5% Triton X-100, 1 mM PMSF (or AEBSF),
10 M Leupeptin, 10 M Pepstatin A, 15 M E-64, 50 M Bestatin, Recombinant
RNasin (Promega) ribonuclease inhibitor 20 units/ml\], prepare fresh and
keep on ice

· Digestion buffer \[50 mM NaCl, 300 mM sucrose, 10 mM Pipes, pH 6.8, 3
mM MgCl~2,\ ~store in aliquots at --20°C. Before use add the following;
0.5% Triton X-100, 1 mM PMSF (or AEBSF), 10 M Leupeptin, 10 M Pepstatin
A, 15 M E-64, 50 M Bestatin, Recombinant RNasin (Promega) ribonuclease
inhibitor 20 units/ml, RQ1 RNase-free*^a^* DNase (Promega) 200-500
units/ml\], prepare fresh and keep at room temperature

· DAPI (Molecular Probes)

*Method*

1\. Remove coverslips from the 75% ethanol solution, flame to burn the
ethanol off and place them into the 35-mm tissue culture dishes or into
the wells of a 6-well tissueculture plate.*^b^*

2\. Plate the cells such that they reach 50-70% confluency at the day of
experiment.*^c^*

3\. Wash the cells twice with PBS at 4°C.^d^

4\. Gently add 3 ml of CSK buffer to each dish and incubate for 10 min
at 4°C.*^e^*

5\. Remove the CSK buffer completely by tilting the dishes and
aspirating the buffer with a glass capillary pipette.

6\. Transfer the coverslip #1 to a new well and fix it by adding 3 ml,
2% paraformaldehyde and incubate for 15 min at room temperature.

7\. Gently add 3 ml of extraction buffer to each dish and incubate for 5
min at 4°C.

8\. Remove the extraction buffer completely by tilting the dishes and
aspirating the buffer with a glass capillary pipette.

9\. Transfer the coverslip #2 to a new well and fix it by adding 3 ml,
2% paraformaldehyde and incubate for 15 min at room temperature.

10\. Gently add 1 ml of digestion buffer and incubate for 30-60 min at
room temperature.*^f^*

11\. Remove the digestion buffer completely by tilting the dishes and
aspirating the buffer with a glass capillary pipette.

12\. Terminate the digestion by gently adding 3 ml of extraction buffer
and incubate for 5 min at 4°C.*^g^*

13\. Remove the extraction buffer and fix coverslip #3, 4, 5 by adding 3
ml, 2% paraformaldehyde, incubate for 15 min at room temperature.

14\. Process all samples for immunofluorescence microscopy.*^h^*

*^a\ ^*It is essential that the DNase I is RNase-free.

*^b^*^\ ^Prepare at least 5 coverslips per sample. The first coverslip
will be extracted only with Triton X-100. The second coverslip will be
extracted with Triton X-100 followed by ammonium sulfate. The third and
fourth coverslips will be extracted with Triton X-100, ammonium sulfate
and digested with DNase I. The fourth coverslip is used as positive
control (see below). The fifth coverslip is not extracted (control). It
is generally convenient to use 6-well tissue culture plates when
multiple samples need to be processed at the same time.

*^c^*^\ ^This also applies to transiently transfected cells.

*^d^*^\ ^If using suspension cells, approximately 10^6^ cells per
extraction step will be adequate. Centrifuge the suspension cells at
650-1000 *g* for 5 min at 4°C between steps 3-13. Resuspend the cells at
the each extraction step in the same volume of buffer as the adherent
cells.

*^e^* Incubate by simply placing the dishes on ice.

*^f\ ^*The duration of digestion and the necessary amount of DNase I
will vary depending upon the cell type used and has to be optimized. A
good starting point, particularly in mouse cell-lines (heterochromatin
forms dense structural domains) is to use 500 units/ml DNase I and three
different incubation times (15, 30 and 60 min) at room temperature.
Monitor the release of chromatin by staining the cells with DAPI for
fluorescence microscopy. DNase I treatment must lead to a complete loss
of DAPI stain. If the digestion is insufficient, use 30 and 60 min
digestions both at 32°C and 37°C.

*^g\ ^*For Western blot analysis, centrifuge the cells as above and
measure the packed-cell volume. In order to extract proteins from the
nuclear matrix, add 2-3 times the packed-cell volume of 5X SDS sample
buffer to a final concentration of 1X SDS. Boil the samples for 5 min.
Vortex or sonicate the samples (depending on the solubility of extract).
Centrifuge the samples in a bench-top centrifuge at 16000 g for 10 min
at room temperature.

*^h\ ^*It is very important to verify the integrity of nuclear matrix by
processing coverslip #4 for indirect immunofluorescence microscopy. Use
an antibody directed against one of the well-known matrix-associated
proteins (e.g. NuMA). The hnRNPA1 and histone H1 proteins could be used
as the negative controls for the detergent and high-salt extraction
steps respectively.