Proteins of different solubility were further extracted from brains in buffers of increasing stringency.
For insolubility assessment, four brain samples for each age and genotype underwent sequential extraction in buffers of increasing stringency, based on a modified protocol previously described [ 43].
All brain samples were homogenized in buffer consisting of 0.05 M Tris HCl, 0.15 M NaCl, 0.1% Nonidet 40, 0.5 M phenylmethylsulfonyl fluoride, 50 mg/mL aprotinin, 10 mg/mL leupeptin, 50 mg/mL pepstatin, 4 mM sodium orthovanadate, 10 mM sodium fluoride, and 10 mM sodium pyrophosphate.
Brains were fixed overnight in buffered 2% paraformaldehyde at 4°C.
After washing with buffer B, membrane pellets were resuspended in 50 µl of rat brain cytosol (10 mg/ml) prepared in buffer A containing protease inhibitors.
This was achieved by incubation of the brain section in blocking buffer containing 1∶500 dilution of AP conjugated anti-DIG antibody (Boehringer Mannheim) follow by the incubation in the detection buffer containing NBT, BCIP and levamisole.
Tissue samples were gently thawed, dissected into ∼1 mm3 fragments, washed with cold PBS and homogenized in 5 volumes of cold brain lysis buffer (BLB; 150 mM NaCl, 50 mM HEPES-KOH, pH 7.3, 10% glycerol, 1 mM EDTA, 5 mM EGTA, 0.5% NP-40, 2.5 mM DTT and 1 mM PMSF) supplemented with RNAse, phosphatase and protease inhibitors as described above.
One hundred microliters of brain homogenate in HS buffer were sonicated for 5 min using a water-bath sonicator and then centrifuged at 100,000 × g for 30 min at 4 °C.
Brains were removed and stored in buffered formalin.
Proteins were extracted from brain tissue in a lysis buffer comprised of 20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1% Nonidet P40, 1 mM EGTA, 1 mM EDTA, and 1% sodium deoxycholate; lysate protein was quantified using a Micro BCA assay reagent kit (Pierce, Rockford IL) as described previously [ 2].
