Application of Zymographic Methods to Study Matrix Enzymes Following Traumatic Brain Injury - Animal Models of Acute Neurological Injuries: Injury and Mechanistic Assessments - page 184

2. Tissue Protein Extraction Reagent Thermo Scientifi c (T-PER)
(Thermo Scientifi c, Rockford, IL, #78510)
3. Pierce BCA Protein Assay Kit (Thermo Scientifi c, #23225)

RIPA Lysis Buffer (Millipore, Billerica, MA, #20-188) 2.1.3. Extraction of Corpus

Callosum and Cortex

2.2. Gel
Electrophoresis
and Incubation

1. Novex 10% Zymogram Gelatin Gels (10-well, 0.1% gelatin,
Tris-Glycine, Invitrogen, Carlsbad, CA, #EC6175)
2. Tris-Glycine SDS Running Buffer (10×) (Invitrogen, #LC2675)
3. Tris-Glycine SDS Sample Buffer (2×) (Invitrogen, #LC2676) 2.2.1. Gelatin Gels

1. Zymography Renaturing Buffer (Invitrogen, #LC2670)
2. Zymography Developing Buffer (10×) (Invitrogen, #LC2671)

2.2.2. Gel Incubation

2.3. Visualization
of Lysis

1. Brilliant Blue R (Sigma-Aldrich, #B7920-50G)
2. Destain solution 1: Methanol:glacial acetic acid:nanopure
water (4:1:5, v:v:v)
3. Destain solution 2: Methanol:glacial acetic acid:nanopure
water (0.7:1:8.3, v:v:v)

2.4. Measurement
of Signal

1. G:Box ChemiHR System (SynGene)
2. GeneSnap Software (SynGene)

2.5. Experimental
Design

1. MMP-2 Standard (Millipore, Billerica, MA, #CC071); proen-
zyme = 68 kDa nonreduced and active (59-62 kDa)
2. Purifi ed human MMP-9 (Millipore, #CC079); proen-
zyme = 88 kDa nonreduced and mature active form = 82 kDa
3. N -ethylmaleimide (NEM) (Sigma-Aldrich, St.Louis, MO,
#E3876)
4. Phenylmethyl sulfonyl fl uoride (PMSF) (Sigma-Aldrich, #P7626)

2.5.1. Standards
and Inhibitors

p -aminophenylmercuric acetate (APMA; Sigma-Aldrich, #A-9563) 2.5.2. Activation

of Pro-MMPs

2.5.3. Drug Manipulations

1. MK-801 (Sigma-Aldrich, #M-107)
2. Minocycline Hydrochloride (Sigma-Aldrich, #286710)

3. Methods
Description

Under general anesthesia (4% isofl urane in carrier 70% N 2 O, 30%
O 2 ), animals are decapitated, the brain rapidly removed, and
regions of interest dissected on an iced platform (Note 1).
Depending upon desired sample site, the brain may be blocked

3.1. Sample
Preparation

3.1.1. Tissue Dissection