Biology Reference
In-Depth Information
Fig. 4. Effect of therapeutic intervention on gelatinolytic profi le. In vivo MK-801 administration
(1.0 mg/kg, i.p., twice daily for 2 days post injury) inhibits both pro MMP-2 (68 kDa) and
MMP-9 activity in T-PER extracts of hippocampus (
a
), as compared to the TBI + BEC vehicle
control. Graph in (
b
) shows signifi cant (
p
< 0.01) reduction in hippocampal gelatinolytic
activity, expressed as a percent of vehicle control.
4. Notes
1. MMPs can be extracted directly from fresh tissue or frozen in
liquid nitrogen and extracted at a later date. This should be
decided based upon the design of the experiment. If small
defi ned regions are targeted, pooling of freshly dissected tissue
into a single frozen sample may be preferable (see Table
1
).
2. Exceptionally high MMP-2 and MMP-9 signal can result from
an injury sample where there is considerable surrounding hem-
orrhage (Fig.
1
). Clean the tissue so that it is free from con-
taminating blood. Such blood contamination can artifi cially
enhance brain tissue signal on zymograms and confound inter-
pretation of results.
3. Inclusion of any necrotic tissue at a lesion site should also be
avoided. Such samples also produce very high activity and, in
most cases, represent unusable outlier data.
4.1. Tissue Dissection
4.2. Protein Extraction
4. MMPs can be extracted from tissue with a variety of buffers
other than T-PER and RIPA. In order to use samples in multiple
endpoint assays, such as Western blots or commercial activity
assays, it may be necessary to use different extraction buffers.
Therefore, we have used RIPA buffer for extraction of activity
in the corpus callosum and the cortex and T-PER or Brij/
Triton in the extractions of the hippocampus (see Fig.
5
).
5. Hippocampal protein extraction for gelatin zymography can
also be accomplished using a Brij/Triton extraction buffer.
Hippocampi are extracted on ice, in Brij/Triton extraction
Search WWH ::
Custom Search