Dilactate form

Conforming to our earlier studies, we performed immunofluorescence labeling, quantification and microscopy of pilins on the cell surface of the selected derivatives (Rasinkangas et al., 2014 (link)). SpaA and SpaC antisera were used as primary labeling agent, followed by detection via secondary labeling with Alexa Fluor 488-labeled goat anti-rabbit antibody (Invitrogen). DAPI (1:1000 dilution, Dilactate form, Thermo Fisher Scientific) was used to stain and thus quantify all cells present in a sample, thus enabling normalization of the fluorescence intensity results for SpaA and SpaC. Fluorescence intensities of each stain were measured separately using a Victor3 1460 multilabel counter (Perkin Elmer). Normalization was performed by dividing the obtained fluorescence intensity values by the corresponding DAPI intensity for each strain. Both quantitative and qualitative data from a representative experiment is shown.

Immunofluorescence labeling, quantification, and microscopy of pilins on the surface of cells were performed essentially as described previously in Rasinkangas et al. (2014 (link)). Initial labeling was performed using SpaA and SpaC antisera followed by secondary labeling with Alexa Fluor 488-labeled goat anti-rabbit antibody (Invitrogen). To normalize the fluorescence intensity for SpaA and SpaC to the total quantity of cells in the samples, chromosomal DNA of the cells was labeled with 1:1000-diluted 4′,6-diamidino-2-phenylindole (DAPI, Dilactate-form, Thermo Fisher Scientific) at the same time with the secondary antibody labeling. Fluorescence intensities of freshly labeled samples were measured with a Victor3 1460 multilabel counter (Perkin Elmer) using separate measurement programs for each dye. The fluorescence intensity results from the immunofluorescence labeling were divided by the corresponding DAPI intensity to obtain a normalized fluorescence value for each strain.