Goat anti mouse conjugated with alexa fluor 488

Immunofluorescence was performed as previously reported with some modifications [27 (link)]. Citrate buffer-based heat-induced antigen retrieval was performed with brain hippocampal slices at 9–100 °C for 20 min. After 10 min of drying, slides were rinsed in PBS, including 0.1% Tween 20 (PBST), and immersed with blocking solution containing 5% normal goat serum and 0.1% TritonX-100 in PBST for 1 h at room temperature (RT). Then, the sections were incubated with primary antibodies diluted in blocking solution at 4 °C in a humidified chamber. The next day, the slides were exposed to room temperature for 1 h, washed in PBST, and incubated with a secondary antibody, goat-anti mouse conjugated with Alexa fluor 488 or rabbit conjugated with Alexa fluor 594 (Thermo Fisher scientific, Waltham, MA, USA) for 90 min at RT. After washing the slides, 4′,6′-diamidino-2-phenylindole (DAPI) was used for 10 min, and sections were mounted under the coverslips. The target protein of interest was detected by confocal laser-scanning microscopy (FV 1000MPE, Fluoview, Olympus, Tokyo, Japan).

Genistein (99% purity; #446–72-0) was purchased from Pharmaceutical Research Institute, Warsaw, Poland). It was dissolved in DMSO at stock concentrations of 30, 60, and 100 mM, and stored at -20 °C. The following antibodies were used: monoclonal mouse anti-human huntingtin clone mEM48 (#MAB5374 for immunofluorescence), and clone 1HU-4C8 (#MAB2166 for Western-blotting) (Sigma-Aldrich, city, Munich, Germany); goat anti-mouse conjugated with Alexa Fluor 488 (#A-11001, Thermo Fisher Scientific, city, Waltham, Massachusetts, USA); anti-β-actin conjugated with horse radish peroxidase (#A3854, Sigma Aldrich, city, Munich, Germany).