Biology Reference
In-Depth Information
11. 100% Ethanol (absolute ethanol): 30
12. Xylene: 30
13. Xylene: 3¢ = time required to coverslip slides in Eukitt.
″
Staining Protocol for Fixed Tissue
1. 1:1 Ethanol-chloroform 100°: 30¢. This step removes lipids
and facilitates cells thionin penetration.
2. 1:1 Ethanol-chloroform 100°: 30¢. This step removes lipids
and facilitates cells thionin penetration.
3. 70% Ethanol: 2¢
4. Distilled H
2
O: 2¢
5. Thionin Stain: 2¢
6. Distilled H
2
O: 2¢
7. 70% Ethanol: 2¢
8. 96% Ethanol: 2¢
9. 96% Ethanol + acetic acid (2 mL in 250 mL): 2¢. This step
improves signal differentiation.
10. 100% Ethanol: 2¢
11. 100% Ethanol: 2¢
12. Xylene: 2¢
13. Xylene: time required to coverslip slides in Eukitt.
After staining and drying, the slides are scanned with high resolu-
tion. The infarct volume can be then delineated under ImageJ
(Rasband W, National Institute of Health, USA). The infarcted
tissue is manually delineated on each slide as is the volume of
healthy tissue of each hemisphere from the beginning to the end of
the lesion (Fig.
4
).
The infarct volume can be calculated multiplying the sum of
the delineated surfaces by the inter-slide spaces. To this purpose
different equations can be used:
3.4 Analysis and
Considerations on the
Use of Animal Models
of MCAO to Test the
Neuroprotective
Effects of EPO
3.4.1
Analysis
Infarctus volume (mm
3
) = (contralateral hemisphere volume × infarct
volume)/total ipsilateral volume
This formula includes edema correction.
Or:
Infarct volume (mm
3
) = (healthy contralateral hemisphere volume −
healthy ipsilateral hemisphere volume)
We described above two procedures to induce experimental isch-
emia. Whereas, the model of intraluminal filament occlusion and
the model of permanent electrocoagulation are widely used in the
literature to study the pathophysiology of stroke, they are also per-
tinent models to test the efficiency of therapeutic strategies.
3.4.2 Use of Animal
Models of MCAO to Test
the Neuroprotective Effects
of EPO
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