Zen software v2010

Endogenous amyloids from fractionated or unfractionated SP were visualized by staining samples with Proteostat and/or pFTAA, using approaches previously described (Usmani et al., 2014 (link)). Briefly, fractionated or unfractionated SP were incubated with Proteostat and/or pFTAA for 15 min at room temperature, and then transferred into an Ibidi Chamber Slide (#80826 from GmbH). Stained samples were imaged on a Zeiss LSM710 AxioObserver confocal microscope equipped with a Plan-Apochromat 63/1.40 oil objective lens and Zen-Software v2010 (Zeiss, Germany). Spermatozoa were simultaneously imaged by DIC using transmitted light detector (T-PMT) and appropriate condenser settings. Where indicated, spermatozoa were additionally visualized by staining with Hoechst 33342.

The infected untreated and treated cells were analyzed by flow cytometry and confocal microscopy to determine the percentage of infected cells and viral protein localization. The samples were processed as previously reported by Soto-Acosta et al.^{14 (link)}. Mouse anti-Env 4G2 monoclonal antibody (MAB10216) was used to determine the percentage of infected cells by flow cytometry. The 4G2 antibody was also used with rabbit polyclonal antiNS4A antibody (GeneTex) to determine viral proteins' colocalization in replication complexes by confocal microscopy. As secondary antibodies, a goat anti-mouse Alexa Fluor 488, a goat anti-mouse Alexa Flour 405 and a goat anti-rabbit Alexa Flour 555 were used (Life Technologies). The nuclei were counterstained with Hoechst 33342 (Life Technologies). The Flow cytometry was performed in a BD LSR Fortessa, and the data were analyzed using the FlowJo v. 10 software. The infected cells were observed in a Zeiss LSM700 laser confocal microscopy to determine colocalization ratios between viral proteins, and images were analyzed with ZEN software v. 2010. Three independent experiments in duplicate were performed to determine the percentage of infected cells, and two independent experiments duplicated for the viral protein localization.