Application of Zymographic Methods to Study Matrix Enzymes Following Traumatic Brain Injury - Animal Models of Acute Neurological Injuries: Injury and Mechanistic Assessments - page 187

Buffer for 30 min at RT while on a rocker platform. Afterward,
gels are washed with 100 ml Development Buffer for 30 min at RT
with rocking. Finally, gels are incubated with 150 ml Development
Buffer in a modifi ed protocol for 6 days at 37°C without rocking
(Note 10).

3.3. Visualization
of Lysis

Following incubation, the gel is washed three times in 100 ml of
nanopure water followed by staining in Brilliant Blue Dye. The
Brilliant Blue Dye (0.5%) is prepared in destain solution 1, mixed,
and allowed to sit at RT so that the undissolved dye remains on the
bottom. Before use, the dye solution is removed carefully without
disturbing any sediment. Lytic activity is then visualized by incuba-
tion with 0.5% Brilliant Blue Dye, 100 ml, for 30 min on a rocking
platform. The staining step is followed by destaining in 100 ml of
destain solution 1 for 30 min with gentle rocking. It is then placed
in 100 ml of destain solution 2 for 30 min while rocking gently,
taking care not to excessively destain (Note 11).

3.4. Measurement
of Signal

Gels are imaged digitally by the G Box System (Synoptics). The gel
is placed directly on the transilluminator box, the image is captured,
and the background light is subtracted by neutral fi elding adjust-
ment (Note 12). The clear lytic bands are identifi ed and signal is
captured by the G Box System. Data is then analyzed densitometri-
cally with Gene Tools software. Results are expressed as percent
change relative to sham-injured controls.

3.5. Experimental
Design

Purifi ed human MMP-2 (0.06 ng) and MMP-9 (0.01 ng) enzymes
are run as standard positive controls (Fig. 2 and Fig. 3 ). Signal
specifi city for MMP is confi rmed by methodological controls
showing that Ca++- and Zn++-induced lysis is eliminated by the
addition of EDTA, a divalent cation chelator, and not affected by
the serine protease inhibitor PMSF or the cysteine protease inhibitor
NEM (Fig. 3 ). The PMSF and NEM solutions are prepared just
before use. A 0.5 M EDTA stock is prepared in nanopure H 2 O, pH
7.8, and brought to a fi nal concentration of 10 mM in developing
buffer. Zymogram Development Buffer contains 5 mM CaCl 2 ;
therefore, EDTA must be added in molar excess of the calcium
chloride. For PMSF, a 0.5 M stock in DMSO is mixed and added
to developing buffer to give a fi nal concentration of 2 mM PMSF.
Finally, a 1 M NEM stock is prepared in ethanol, and also added to
developing buffer to give a fi nal concentration of 10 mM NEM.

3.5.1. Controls with
Inhibition of Lysis

Confi rmation of pro-MMP shift to its active form can be tested by
APMA exposure (see again Fig. 3 ). The APMA stock solution of
10 mM is prepared in 0.05 N NaOH and then diluted to 1 mM
using a Tris-HCl buffer (50 mM Tris-HCl, pH 7.4, 0.01 M CaCl 2 ,
and 0.04% sodium azide). A 20-

3.5.2. Controls for
Pro-MMP Activation

g aliquot of extracted supernatant
from both sham- and brain-injured animals is incubated with 1 mM
APMA for up to 8 h at 37°C. The samples are then subjected to

μ