Biomedical Engineering Reference
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Fig. 4. Example data of immunofluorescence microscopy showing co-localization of
transcriptional factor with mutant Htt inclusion in R6/2 mouse brain. 10
m
m coronal
sections prepared from frozen brain of a 12-week-old R6/2 mouse were fixed and
stained with anti-NF-YA (sc-10779; SantaCruz) rabbit antibody together with anti-
ubiquitin mouse antibody. Nuclei were detected with DAPI. Nuclear inclusions positive
for ubiquitin were stained by NF-YA antibody in cortex and striatum.
13. Wash the sections with TBST for 10 min.
14. Cover the sections by coverslip with VECTASHIELD
Mounting Medium.
15. Analyze the sections with confocal microscope system or flu-
orescence microscope (see Fig.
4
).
3.8. Electrophoretic
Mobility Shift Assay
EMSA is one way to examine the DNA binding ability of tran-
scriptional factors. By EMSA, you can examine whether the
sequestration of transcriptional factor leads to reduction of its
function in vivo. In the following experiment, isolated striatum
and cortex from R6/2 mouse brain are used because these regions
are severely affected in HD mouse models and HD patients.
Before doing this experiment, you have to check the expression
level of your transcriptional factor in HD model mouse brain by
RT-PCR using standard methods. This is very important because
it is possible that reduction of DNA binding of the transcriptional
factor is just caused by its reduced expression but not by seques-
tration. The following methods are modifications of the ones
established in Dr. Morimoto's laboratory in Northwestern
University (Chicago, USA) (
15
). The original methods can be
found on the lab's homepage (
http://www.biochem.northwest-
1. Mix 1 ml of sense oligonucleotide (10 pmol/ml) with 1 ml of
g[
32
P]-ATP (6,000 Ci/nmol), 1 ml T4 DNA kinase, 5 ml of
10× kinase buffer and 42 ml of H
2
O.
2. Incubate at 37°C for 0.5-2 h.
3.9. Probe Preparation
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