M . Maruyama et al. Brain Research 887 2000 350 –358
351
microglias [24]. Moreover analysis performed in ATF2 chased from CLEA Japan Inc. Under deep anesthesia with
null mice has indicated that ATF2 plays a critical role in an intraperitoneal injection of sodium pentobarbital, mice
the hippocampal CA3 [20]. These results suggest that p38 were perfused through the left cardiac ventricle with saline
may play a critical part in maintenance of central nervous followed by ice-cold 4 paraformaldehyde in 0.1 M
system CNS through the determination of survival and phosphate buffered saline PBS, pH 7.4. The brain was
or death in response to extracellular stimuli. then removed and immersion fixed in the same fixative for
Here we report the localization of p38 in the adult 12 h at 48C. For paraffin sections, the brain was dehydrated
mouse brain as a first step to understanding its physiologi- in graded ethanol, embedded in paraffin, and sectioned at 4
cal functions in the CNS. mm thickness. For cryostat sections, the brain was im-
mersed in the series of 5, 15 and 30 sucrose solutions in PBS for cryoprotection, frozen in Tissue Tek O.C.T.
2. Material and methods compound Miles Inc., and sectioned at 10 mm thickness.
For immunoperoxidase labeling, sections were blocked 2.1. Antibodies
by incubation with 10 normal horse serum in PBS for 30 min followed by several washes with PBS. They were then
A polyclonal anti-p38 antibody was raised by immuniz- incubated with a primary antibody in PBS for 1 h followed
ing rabbits
with an
affinity purified
recombinant by several washes with PBS containing 0.05 Triton
glutathione-S-transferase-p38a GST-p38 [22]. The anti- X-100
PBST. Then
sections were
incubated in
GST-p38 serum was repeatedly applied to GST-c-Jun [11] biotinylated secondary antibody for 1 h followed by
column to remove antibodies against GST and bacterial several washes with PBS. They were then incubated in
proteins. Then, partially purified serum was multi-step ABC reagent followed by several washes with PBS. Bound
affinity purified by binding to GST-p38 column. Mono- peroxidase was visualized by incubating sections with
clonal antibodies against neurofilament 68, 160, 200 and 0.05 diaminobenzidine and 0.01 H O
in 50 mM
2 2
CNPase were purchased from Sigma-Aldrich, Japan K. K. Tris–HCl, pH 7.4. Control sections were incubated with
Alexa 488– and Alexa 594–conjugated second antibodies pre-immune serum. All incubations were performed at
were purchased from Molecular Probes. Biotinylated sec- room temperature.
ondary antibodies and ABC reagents were purchased from For immunofluorescence double labeling, sections were
Vector Laboratories. blocked by incubating with 10 normal goat serum in
PBS for 30 min followed by several washes with PBS. 2.2. Immunoblot analysis
Then they were incubated with a primary antibody in PBS with 2 normal goat serum for 1 or 3 h followed by
Dissected brain tissues for immunoblotting were ob- several washes with PBST. Alexa 488– and Alexa 594–
tained from adult mouse and sonicated in buffer containing conjugated second antibodies were diluted 2000-fold and
50 mM Hepes–KOH pH 7.4, 150 mM NaCl, 5 mM incubated for 1 h in PBS containing 2 goat serum. After
EDTA, 5 mM EGTA, 20 mM NaF, 1 mM dithiothreitol, 1 extensive washing with PBS, sections were examined by
mM phenylmethylsulfonylfluoride, 1 mM sodium ortho- using fluorescent microscopy Olympus. Control sections
vanadate, 1 mM p-nitrophenylphosphate, 20 mM b- were incubated either with pre-immune serum or antibody
glycerophosphate and 1 Triton X-100. Then 100 mg of preabsorbed with GST-p38.
protein was loaded in each lane after boiling for 3 min in The terminology of brain areas was mainly after Paxinos
sample buffer containing sodium dodecyl sulfate SDS. and Watson [18] and Franklin and Paxinos [6]
The tissue samples were resolved by 12 SDS–poly- acrylamide gel electrophoresis and transferred onto Im-
mobilon Millipore. The filters were blocked for 1 h in 5 non-fat dried milk in buffer containing 10 mM Tris–HCl
3. Results
