***In vivo labeling of newly-synthesized RNA with 5-Bromouridine
5**'**-Triphosphate and immunodetection of transcription sites by
microscopy***

*Equipment and reagents*

· Tissue culture dishes (35 mm)

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

· Phosphate-buffered saline (PBS)

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

· Microinjection buffer (100 mM BrUTP (Sigma), 140 mM KCl, 2 mM Pipes,
pH 7.4), store in aliquots at --20°C

· Monoclonal mouse antibodies to bromodeoxyuridine; clone BR-3 (Caltag)
or clone BMC-9318 (Roche Molecular Biochemical) or clone Bu-33 (Sigma)

· Actinomycin D (Calbiochem) and a-amanitin (Sigma)

· RNase A (Sigma)

· DAPI Nucleic Acid Stain (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.

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

3\. Take the first dish out of tissue culture incubator and place onto
the stage of fluorescence microscope equipped with a microinjection
system.*^b^*

4\. Inject the injection buffer into the cytoplasm of cells.*^c^*

5\. Place the dish back in the tissue culture incubator and incubate 5
min at 37°C.*^d^*

6\. Take the dish out of tissue culture incubator and gently add 1 ml of
PBS while lowering the tilted dish.

7\. Remove the PBS completely by tilting the dish and aspirating the PBS
with a glass capillary pipette.

8\. Fix the cells by adding 3 ml of 2% paraformaldehyde and incubate for
15 min at room temperature.

9.* *Remove the paraformaldehyde by aspirating it with a glass capillary
pipette.

10\. Process all of the samples for immunofluorescence microscop*y.*

*^a^* This also applies to transiently transfected cells.

*^b^* Keep the cells in the growth medium. The growth media change or
PBS wash is not necessary at this stage.

*^c^* Inject always less than 5% of total cell volume. One indication
that the cells are injected with more volume than necessary is the
apparent change in cell morphology. Particularly, the edges of cells
become uneven and cells lose their round shape. This can also be
observed easily by staining the cells with DAPI after the
paraformaldehyde fixation (step 8). To optimize the microinjection
technique, replace the BrUTP with a fluorescent dye such as Rhodamine in
the injection buffer and inject different volumes of buffer into
cytoplasm, while observing the cells by fluorescence microscopy.

*^d\ ^*The duration of incubation may vary depending upon the cell type
used and has to be optimized. A good starting point is to use four
different incubation times (5,15, 30 and 60 min). Once the transcription
sites are labeled successfully on a regular basis, it is important to
further optimize the labeling period until transcription site labeling
is achieved with minimum labeling time.

· Several controls must be performed to verify whether the observed
sites are genuine transcription sites:

\(a\) Do an experiment with UTP in the transcription and microinjection
buffers instead of BrUTP. There should be no labeling detected by
indirect immunofluorescence microscopy demonstrating the specificity of
the antibody.

\(b\) Do a labeling with transcription and microinjection buffers
containing 1-10 g/ml -amanitin or 5-10 g/ml actinomycin D. If used in
these concentrations, -amanitin specifically inhibits the transcription
by RNA polymerase (RNA pol) II. Thus, only the nucleolar transcription
sites, but not the nuclear transcription sites should be detected.
Actinomycin D at the above concentration, inhibits transcription by all
three RNA polymerases.

· If no labeling is observed

\(a\) Repeat the experiment by using either one of the other mouse
monoclonal antibodies.

\(b\) Increase the duration of labeling.

\(c\) Do the labeling at 37°C.

\(d\) Increase the concentration of BrUTP to 1 mM.

\(e\) Optimize the concentration of detergent.

\(f\) Use a biotin-streptavidin fluorescent signal enhancement system
(e.g. Biotin-conjugated anti-mouse IgG and Texas Red dye-conjugated
Streptavidin) in indirect IF. These options can be used individually or
in combination.