Antigen and antibody preparation
Fibril antigens were prepared by stirring 2 mg/ml Aβ42 peptide in 50% HFIP/H2O, 0.02% sodium azide for 7 days. Afterwards, the HFIP was evaporated under a stream of nitrogen and the sample was stirred for an additional 7 days and dialyzed against PBS (molecular weight cut off 10,000 Da). The resulting fibrils were checked by EM and the purity was confirmed by the absence of oligomers using anti-oligomer antibody. The same protocol was used to prepare IAPP fibrils. Aβ and IAPP oligomer mimics were prepared as previously described  The synthesis of IAPP C-terminal thioester analog lacking cysteine residues (KANTATAATQRLANFLVHSSNNFGAILSSTNVGSNTY-SR), was also carried out according to the methods described . The antigens were each used to immunize two New Zealand white rabbits (Pacific Immunology Corp., Ramona, CA, 92065) according to protocols approved by IACUC. Each rabbit immunized with 500 μl of antigen in complete Freund's adjuvant (CFA), and then boosted twice at four week intervals with 500 μl of antigen in Incomplete Freund's Adjuvant (IFA).
Fibril and oligomer preparation
Aβ fibrils and fibrillar oligomers were prepared by dissolving 0.3 mg of lyophilized Aβ42 in 150 ul of hexafluoro-2-propanol (HFIP) for 10–20 minutes at room temperature. The resulting Aβ solution was added to DD H2O in a siliconized Eppendorf tube to 80 uM concentration. After 10–20 min incubation at room temperature, the samples were centrifuged for 15 min. at 14,000 × G and the supernatant fraction (pH 2.8–3.5) was transferred to a new siliconized tube and subjected to a gentle stream of N2 for 10 min to evaporate the HFIP. The sample was then stirred at 500 RPM using a Teflon coated micro stir bar for 24 hours at 22°C. This method was originally reported for preparing A11 positive prefibrillar oligomers , but more recent work indicates that it also produces fibrillar oligomers that are OC positive. Fibrils were separated from fibrillar Aβ42 oligomers by centrifuged at 100,000 × G for 1 hour at 4°C. The supernatant containing fibrillar oligomers and pellet fraction containing fibrils were separated and collected. The pellets were resuspended in an equal volume of H2O. Alternatively, 1 mg of lyophilized Aβ42 was dissolved in 200 μl of DMSO and incubated at room temperature for 10–15 minutes to form fibrillar oligomers. The fibrillar oligomers in DMSO were fractionated according to size using a TSK-GEL SuperSW2000 column (Tosoh Bioscience LLC) in 10 mM Phosphate, pH 7.4 at a flow rate of 0.3 ml/min.
Prefibrillar oligomers that are OC negative were prepared as previously described . Aβ42 stock solutions (2 mM) were prepared by dissolving the lyophilized peptide in 100 mM NaOH followed by water bath sonication for 30 s. The oligomerization reaction was initiated by diluting the stock solution in phosphate buffered saline (PBS), pH = 7.4, 0.02% sodium azide (45 μM final Aβ42 concentration, final pH = 7.4) and incubated at room temperature for up to 15 days. Oligomer formation was monitored by dot blot with A11 and OC polyclonals.
ADDLs were prepared according to Lambert et al . Aβ42 peptide was initially incubated in HFIP at room temperature for 1 hour. The HFIP was evaporated under a gentle stream of N2. The remaining Aβ42 was dissolved in anhydrous DMSO at 5 mM, diluted into cold phenol red-free F12 medium and aged at 4°C for 24 hours. The final peptide concentration was 80 μM.
Q36 fibrils were prepared by dissolving Q36 in 10 mM HEPES/100 mM NaCl at a concentration of 45 μM and incubating with stirring at 25 C for 6 days. Fibril formation was confirmed by EM and ThT. Q36 oligomers were prepared by dissolving Q36 in 1:1 (vol:vol) TFA:HFIP at 2 mM and diluting it to 45 μM in PBS and incubating with stirring for 6 days. Oligomer formation was confirmed with A11 antibody.
ELISA, dot blot, Western blot assays and Immunohistochemistry
ELISA, dot blot and western blots assays were done as previously described . Brain tissue from autopsy cases was provided by the University of California Alzheimer's Disease Research Center and the Institute for Brain Aging & Dementia Brain Tissue Resource. For single-labeling experiments, 50 μm 4% paraformaldehyde-fixed sections containing the hippocampus and entorhinal cortex or the midfrontal cortex from an AD case (Case #1 – 65 year old female, 7.4 hour post mortem interval, MMSE = 21 15 months prior to death with a Braak & Braak stage of VI) were washed with 0.1 M Tris-buffered saline (TBS), pH 7.5, and then pretreated with 3% hydrogen peroxide in 10% methanol to block endogenous peroxidase activity. Sections were subsequently washed in TBS with 0.1% Triton X-100 (TBS-A) and then blocked for thirty minutes in TBS-A with 3% bovine serum albumin (TBS-B). In a series of preliminary studies, the effects of different concentrations of formic acid during a pretreatment phase was determined to have little effect on the extent or intensity of OC labeling of fixed tissue (data not shown) and thus this step was eliminated from the protocol. Sections were incubated overnight at room temperature in OC at a dilution of 1:10,000. Following two washes with TBS-A and a wash in TBS-B, sections were incubated in either goat anti-mouse or goat anti-rabbit biotinylated anti-IgG and then in avidin biotin complex (ABC) (Vector Laboratories, Burlingame, CA, USA). Antibodies were visualized using 3,3'-diaminobenzidine (DAB, Vector Laboratories). For quantification experiments, the frontal cortex from all cases used for quantification was immunostained in single experiments to reduce variability in the image analysis procedures and all immunohistochemical procedures were identical for the OC antibody. To determine the specificity of the OC antibody, IgG purified serum at a concentration of 1:10,000 was preincubated in 10× and 100× concentration of OC peptide for 2 hours at RT. Subsequently, a section from the midfrontal cortex was incubated in the OC antibody/peptide solution and processed for immunohistochemistry. Sections were counterstained with cresyl violet.
Mouse strain and tissue collection
Hemizygous human IAPP transgenic mice (FVB-tg(IAPP)6Jdm/-Avy/A) were characterized in details elsewhere . For this study we used 24 wk old mice. Before collection of mouse pancreata, the heart was perfused with 10 ml of 4% paraformaldehyde. The pancreas was dissected in cold PBS, fat and lymph nodes trimmed, the pancreas weighed, fixed in 4% paraformaldehyde at 4°C for 24 h then frozen in OCT.
Immunofluorescence and confocal microscopy
An additional AD case was used for confocal studies. Case #2 was a 95 year old female with a post mortem interval of 5.5 hours and a MMSE score of 12, 6.3 months prior to death. Sections containing the midfrontal cortex were incubated in OC overnight (1: 5,000) and positive labeling was detected using anti-rabbit IgG Alexa Fluor 568 (Molecular Probes, Eugene, OR; 1:200). Alexa Fluoro 488 (Molecular Probes, Eugene, OR; 1:200) was subsequently used to visualize LOC (1:5,000). The order of the primary antibodies was also reversed in additional experiments and the results were similar. Because both antibodies were raised in the same species (rabbit) an intervening formaldehyde treatment was used between the first and second immunolabel . Sections were allowed to dry on slides prior to rehydration and coverslipping with VectaShield (Vector Laboratories, Burlingame, CA, USA). Confocal images were collected on an Olympus IX70 inverted microscope and a BioRad Radiance 2000 Laser Scanning System using a 40, 60 or 100× objective for image analysis. Each antibody label was excited and scanned using lambda strobing and barrier filters at 510 and 480 nm. A z-series scan at either 0.5 or 1 μm intervals was captured to determine the spatial colocalization characteristics of OC with LOC.