Human brain specimens from the temporal cortex, frontal cortex, and cerebellum were obtained from the MA Alzeimer Disease Res. Center brain bank. Controls obtained from Harvard Brain Tissue Resource Center.
Samples were homogenized in Tris-buffer with Triton-X100, sodium chloride, EDTA and protease inhibitor cocktail (Roche) and 2% BSA. Supernatant fluid was used for BACE activity assay and protein. For Ab assays, the cortex was homogenized without detergent and the pellet was treated in a solution of formic acid. The resulting supernatant was neutralized with Tris buffer and used in an assay of formic acid-extractable Ab species.
BACE protein assay used a sandwich ELISA with monoclonal anti-BACE (1:4000, Chemicon, No. MAB5308) and polyclonal anti-BACE (1:750, ABR, No. PA1-756). Fluorometric readings were taken with 320-nm excitation filter and 400-nm emission filter after the addition of HRP substrate (QuantaBlu).
BACE enzymatic assay also used antibody MAB5308 but with a fluorogenic substrate for beta-secretase, either MOCA-DNP (Peptides International) or DABCYL (Calbiochem).
For immunoprecipitation the homogenates were rehomogenized in extraction buffer and the supernatant was incubated with protein G-Sepharose (Pierce)to remove non-specific protein binding. After centrifugation the supernatant was incubated with antibody MAB5308 or PA1-756 also bound to G-Sepharose. Eluted samples were separated on 10-20% SDS polyacrylamide gel electrophoresis, transfered to PVDF membrane, probed with antibody MAB5308 and developed with chemiluminescence (Perkin-Elmer) for the BACE band.
Ab 40 and 42 were determined by ELISA using mouse monoclonal anti-Amyloid-b 11-28 (BA27, Takeda Chemical Industry) as the the capture antibody and monoclonal anti-Ab 40 or anti-Ab BC05 conjugated to HRP as the detector antibody. Total formic acid amyloid-beta was determined from the total ELISA results for Ab 40 and 42.
ELISA assay for synaptophysin used monoclonal anti-synaptophysin (1;2000, Chemicon) as the capture antibody and biotinylated monoclonal anti-synaptophysin (SY38, 1:2000, Progen) with HRP-streptavidin as detector. Color was developed with HRP and 3,3',5,5'-tetramethylbenzidine (Kirkegaard and Perry).
The following antibodies were used for immunohistochemistry on brain sections (paraformaldehyde fixed, cut on freezing sledge microtome): MAB5308, 1:500; Rabbit polyclonal anti-GFAP (glial fibrillary acidic protein, 1:500, Dako), rabbit polyclonal anti-Amyloid-beta R1282 (1:500, gift of D. Selkoe, Boston, MA) and mouse monoclonal anti-phospho-gammaTG3 (1:10, gift of P. Davies, Bronx, NY).
They were incubated with secondary antibodies biotinylated anti-mouse IgG, Cy-5-linked anti-rabbit IgG (Jackson), BODIPY-FITC anti-rabbit IgG or anti-mouse IgM (Molecular Probes) or Cy-3linked streptavidin (Jackson). Images were captured with a confocal scanning system (MRC 1024, Biorad).
Statistics for BACE activity and protein were analyzed by ANOVA. t-test was used to determine which brain regions were significant and within the brain regions correlation analysis was used to correlate BACE activity and protein with formic-acid Ab or synaptophysin and length of illness (StatView).