Ab9234

Aliquots containing 15–150 ng of protein were spotted in duplicate on a nitrocellulose membrane and blocked for 1 h in TBST containing 1% BSA. Detection was performed by a 2-h incubation with the anti-amyloid fibril OC antibody (AB2286 Merck Millipore, 1:2000 dilution), anti-amyloid oligomer A11 antibody (AB9234 Merck Millipore, 1:2000 dilution) and sera from patients allergic to fish (1:10 dilution) prepared in TBST-1% BSA and a 30-min incubation with horseradish peroxidase-labeled mouse monoclonal B3102E8 anti-human IgE (Abcam, 1:2000 dilution) or goat anti-rabbit IgG (Sigma-Aldrich, 1:5000 dilution). The signal was developed by the ECL-Clarity Western blotting reagent (Bio-Rad) and detected with a ChemiDoc XRS. Quantifications and analysis were performed as before using the signals from gmPV1 as normalization factors.

Aliquots containing 100 ng Ca²⁺-bound monomers and amyloids of Gad m 1 chains were spotted in duplicate on a nitrocellulose membrane. Immunodetection was performed by 1 h of incubation with anti-amyloid fibril OC (AB2286 Merck Millipore, 1/2000 dilution) and anti-amyloid oligomer A11 (AB9234 Merck Millipore, 1/2000 dilution) antibodies, followed by extensive washes and 30 min of incubation with horseradish peroxidase-labeled goat anti-rabbit IgG (1:5000 diluted; Sigma-Aldrich) [25 (link),26 (link)]. ECL Western blotting reagent (BioRad) and a ChemiDoc XRS instrument (BioRad) were used for signal development and detection, respectively.

Immuno-fluorescent labeling of Aβ oligomers in cultured MC65 cells was conducted using the anti amyloid beta antibody listed in the prior section. Briefly, MC65 cells were plated in a 6 well plate in complete media. The cells were allowed to attach for a period of 48 h. The media was then changed to either complete media with Tetracycline or opti MEM without Tetracycline or opti MEM without Tetracycline and with solution of D-520 (20 μM) prepared after six days of shaking. 48 h following the treatments, the cells were fixed in 4% paraforamaldehyde in PBS for 15 min at room temperature. Blocking was done by incubating the cells in 5% BSA in PBS for 1 h at room temperature. The cells were subsequently incubated in anti-amyloid oligomers antibody (AB9234, Millipore) at a 1:1000 dilution at 4 °C overnight. Cells wells were next washed thrice with PBS for five minutes each to remove any unbound primary antibody. The cells were then incubated with anti-mouse alexa fluor 488 at a dilution of 1:500 for 1 h at room temperature. Any unbound secondary antibody was removed by washing the plates thrice with PBS. The cells were then imaged using an Olympus fluorescence microscope at 40X magnification.