Assessments of Reactive Astrogliosis Following CNS Injuries - Animal Models of Acute Neurological Injuries: Injury and Mechanistic Assessments - page 59

2. Phosphate-buffered paraformaldehyde (PFA), need ~250 ml
per adult rat and ~100 ml per adult mouse
a. Warm distilled water (dH 2 O) to 58-60°C on hot plate in
fume hood and maintain temp, but do NOT exceed 60°C.
b. Add 10 M NaOH.
c. Add PFA and stir until dissolved (solution will be clear,
slightly hazy).
d. Add 0.4 M phosphate buffer (PB).
e. pH solution to 7.2.
f. Filter.
g. Chill to 4°C before use.
h. For 1,000 ml 4% PFA:

dH 2 O 750 ml

10 M NaOH 14 drops

Paraformaldehyde 40 g

0.4 M Phosphate buffer 250 ml

i. See Note 2.

2.2. Antibodies
Commonly Used to
Detect Expression of
Intermediate Filaments
in Fixed Tissue

Frequently used and commercially available antibodies for detec-
tion of GFAP or vimentin in fi xed tissue are listed in Table 1 .

2.3. Materials and
Equipment Needed for
Diolistic Labeling
(Adapted from Ref. ( 34 ) )

1. Gold microcarriers (Bio-Rad)
2. Polyvinylpyrrolidone (PVP, Bio-Rad): prepare a 75 mg/ml
solution in ETOH
3. Methylene chloride (Sigma), see Note 3
4. DiI and DiO (Molecular Probes)
5. Helios gene gun (Bio-Rad)
6. Tubing preparation station equipped with tubing cutter and
Tefzel tubing (Bio-Rad)
7. Medical-grade helium and nitrogen gas cylinder (BOC) and
associated regulators (Bio-Rad)

3. Methods

3.1. Tissue Fixation
and Extraction

1. At the desired post-injury time point, humanely euthanized
the experimental animal. See Note 4.
2. Perform a transcardiac perfusion of PBS followed by phos-
phate-buffered PFA, see Note 5.