Immunohistochemical Analysis of Amyloid-FXR1 Colocalization

Conformation-dependent amyloid-specific OC antibodies (AB2286, Merck, Burlington, MA, USA) were used at a dilution of 1:400, and the primary antibodies anti-FXR1 (ab129089, Abcam, Cambridge, UK) were used at a dilution of 1:600. Cryosections were pretreated as described earlier [5 (link)] and incubated with primary antibodies at +4 °C, overnight. After washing to remove unbound antibodies, the sections were incubated with anti-rabbit secondary antibodies conjugated with Alexa Fluor 647 (ab150075, Abcam, Cambridge, UK) at a dilution of 1:1000 for an hour at +37 °C. To visualize nuclei, the slides were counterstained with DAPI. Histological staining of amyloids was performed using 0.1 mg/mL CR solution in 50% ethanol or using 1% Thioflavin S solution in 70% ethanol. Visualization of the results of immunohistochemical analysis and staining with amyloid-specific dyes was performed with a TCS SP5 confocal microscope (Leica Microsystems, Wetzlar, Germany) and “Leica Application Suite X 3.3.0.16799” software. To quantify the colocalization of FXR1 with CR red or Thioflavin S, Pearson’s correlation coefficient was calculated using the coloc2 plugin of FIJI software (http://fiji.sc/Fiji, accessed on 23 February 2020). At least 100 zones on preparations of each animal studied were analyzed to assess the colocalization of CR or Thioflavin S with the FXR1 protein.