Alexa fluor 488 conjugated anti mouse or anti rabbit secondary antibodies

SH-SY5Y cells were seeded on glass coverslips and treated for 15 min with oligomers of various proteins at the listed concentrations in the absence or presence of SQ. After incubation, the cells were washed with PBS and counterstained with 5.0 μg/ml of Alexa Fluor 633-conjugated wheat germ agglutinin (Life Technologies, CA, United States) (Perni et al., 2017 (link)). After washing with PBS, the presence of oligomers was detected with 1:800 diluted mouse monoclonal 6E10 anti-Aβ antibodies (BioLegend, San Diego, CA, United States) or 1:800 rabbit anti-HypF-N antibodies (Primm, Milan, Italy) and subsequently with 1:1,000 diluted Alexa Fluor 488-conjugated anti-mouse or anti-rabbit secondary antibodies (Life Technologies, CA, United States). Fluorescence emission was detected after double excitation at 488 and 633 nm by a scanning confocal microscopy system (Perni et al., 2017 (link)), and three apical sections were projected as a single composite image by superimposition. ImageJ (NIH, Bethesda, MD, United States) and JACOP plugin (rsb.info.nih.gov) software were used to calculate the percentage of colocalization between cell membranes and oligomers.

SH-SY5Y cells were seeded on glass coverslips and treated for 15 min with HypF-N oligomers (6 µM) or Aβ₄₀ oligomers (5 µM) or in the absence or presence of increasing concentrations of trodusquemine (10:1, 3:1, and 1:1 ratios of oligomers to trodusquemine, monomer equivalents). After incubation, the cells were washed with PBS and counterstained with 5.0 µg mL⁻¹ Alexa Fluor 633-conjugated wheat germ agglutinin (Life Technologies, CA, USA)^{22 (link),28 (link)}. After washing with PBS, the presence of Aβ₄₀ or HypF-N oligomers was detected with 1:800 diluted mouse monoclonal 6E10 anti-Aβ antibodies (BioLegend, CA, USA) or 1:800 diluted rabbit monoclonal anti-HypF-N antibodies (Primm, Milan, Italy) and subsequently with 1:1000 diluted Alexa Fluor 488-conjugated anti-mouse or anti-rabbit secondary antibodies (Life Technologies, CA, USA) to recognize the 6E10 and anti-HypF-N primary antibodies, respectively. Fluorescence emission was detected after double excitation at 488 nm and 633 nm by the scanning confocal microscopy system described previously^{22 (link)} and three apical sections were projected as a single composite image by superimposition. The percentages of oligomer colocalization were determined by analyzing regions of interest corresponding to 50–60 cells per condition.