Biochemical and Molecular Biological Assessments of Intracerebral Hemorrhage - Animal Models of Acute Neurological Injuries: Injury and Mechanistic Assessments - page 637

PBS, containing a protease inhibitor cocktail (Roche, Indianapolis,
IN), and homogenized. The homogenates are then centrifuged at
7,500 × g for 20 min at 4°C. TNF-a in the supernatant is determined
using an ELISA kit (Endogen, Woburn, MA, USA) according to
the manufacturer's recommendations and results are expressed as
picogram per gram (pg/g) of brain tissue.

1. Anti-rat TNF-a pre-coated 96-well strip plates are used. Add
50 mL of the Pretreatment buffer to each well of the microtiter
plate.
2. Add 50 mL of the reconstituted standard or diluted samples to
each well in duplicate. Mix by gently tapping the plate several
times.
3. Add 50 mL of Standard Diluent to all wells that do not contain
standards or samples. Cover the plate and incubate for 1 h at
room temperature.
4. Wash the plate three times with Wash Buffer.

2.2.2. Sample Incubation

5. Add 50 mL of Biotinylated Antibody Reagent to each well.
Cover the plate and incubate it at room temperature for 1 h.
6. Wash the plate three times.

2.2.3. Biotinylated Antibody
Reagent Incubation

7. Add 100 mL of Streptavidin-HRP Regent to each well.
8. Cover and incubate the plate at room temperature for 30 min.
9. Wash the plate three times.

2.2.4. Streptavidin-HRP
Reagent Incubation

10. Pipette 100 mL of TMB substrate to each well.
11. Allow the color reaction to develop at room temperature in the
dark for 10 min. The substrate reaction yields a blue solution
that turns yellow when Stop Solution is added.
12. Stop the reaction by adding 100 mL of Stop Solution to each
well.

2.2.5. Substrate Incubation
and Stop Step

13. Measure absorbance on a plate reader set at 450 and 550 nm.
Subtract 550 nm values from 450 nm values to correct for opti-
cal imperfection in the microplate. If an absorbance at 550 nm
is not available, measure the absorbance at 450 nm only. (Note:
Evaluate the plate within 30 min of stopping the reaction.)
14. The standard curve is used to determine rat TNF-a amount in
an unknown sample. Generate the standard curve by plotting
the average absorbance obtained for each standard concentra-
tion on the vertical (Y ) axis versus the corresponding TNF-a
concentration (pg/ml) on the horizontal ( X ) axis.

2.2.6. Absorbance
Measurement