Assessments of Reactive Astrogliosis Following CNS Injuries - Animal Models of Acute Neurological Injuries: Injury and Mechanistic Assessments - page 61

2. Vibratome sectioning:
Affi x tissue to plate or platform of vibratome.

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Submerge tissue in 0.1 M PB.

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Section tissue by passing vibrating knife through specimen.

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Collect tissue in 0.1 M PB in a collection plate (i.e., 24-well

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plate) or directly onto warm (40°C) 1% gelatin-subbed
slides.

3.3. Immunohisto-
chemistry for GFAP
expression

1. Rinse: 3× 10 min in 0.1 M PB on shaker.
2. Perform endogenous peroxidase blocking treatment:
0.5% H 2 O 2 in 0.1 M PB for 30 min
0.5% H

2 O 2 = 800 ml 30% H 2 O 2 in 50 ml 0.1 M PB.
Keep minimal—weakens fl uorescence.

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3. Rinse in 0.1 M PB 3× 5 min.
4. Rinse in 0.1 M PBS 3× 5 min.
5. Block nonspecifi c background with 3% goat serum + 3%
BSA + 0.3% Triton X + 0.05 M. PBS. Incubate 10 min at RT on
the shaker, then 40 min at 37°C, followed by 10 min at RT on
shaker.
25 ml blocking solution example:

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3% GS = 750 ml
3% BSA = 0.75 g
0.3% Triton X = 75 ml
0.1 M PBS = 24.2 ml
6. Rinse 1× 5 min in 0.1 M PBS.
7. Primary antibody: Dilute rabbit anti-GFAP (#Z0334, Dako)
at 1:4,000 titre in a diluent cocktail of 1% GS + 2% BSA + 0.3%
Triton X + 0.05 M PBS. Incubate 30 min at 37°C, then 20-30 h
at 4°C on the shaker.
25 ml Primary antibody diluent example:

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1% GS = 250 ml
2% BSA = 0.5 g
0.3% TX = 75 ml
0.1 M PBS = 24.7 ml
Dako anti-GFAP (1:4,000) = 6.25 ml

8. Rinse in O.1 M PBS 9× 10 min.
9. Secondary antibody: Dilute the Goat FITC anti-rabbit
(#AP307F Chemicon) to 1:400 titre in 1% GS + 2% BSA + 0.3%
TX + 0.05 M PBS. Incubate for 24 h at 4°C on the shaker or
overnight at RT in dark. See Note 7.