Leica dm5500 b epifluorescence microscope

Immunohistochemical staining using appropriate antibodies (Supplementary Table S2) was performed as follows. Tissue sections were mounted on glass slides, dewaxed, rehydrated and submitted to antigen retrieval by boiling for 20 min in a citrate buffer (pH 6). Endogenous peroxidase activity was blocked by a 10 min incubation with 3% hydrogen peroxide. Slides were then washed in PBS and blocked for 30 min in 2% horse serum. Slides were incubated overnight at 4 °C with primary antibody diluted in PBS, 20% blocking buffer. Bound primary antibody was revealed by 30 min incubation with peroxidase-conjugated secondary antibody (ImmPRESS reagent kit, Vector Laboratories) and finally with 3,3′-diaminobenzidine (DAB substrate reagent kit, Vector Laboratories). For immunofluorescence, 4% formaldehyde-fixed embryoid bodies (EB) or cell mixture aggregates (CMA) were submitted to antigen retrieval with citrate buffer (pH 6). Sections were then blocked either in 2% horse serum, and the slides were mounted with the Vectashield 4,6-diamidino-2-phenylindole (DAPI) medium (Vector Laboratories, Newark, CA, USA). Imaging was performed using a Leica DM5500 B epifluorescence microscope (Leica Microsystems, Wetzlar, Germany) equipped with a CoolSNAP HQ² camera (Photometrics) and Leica MMAF software (Metamorph). Images were processed with Image J software.

Cell growth was quantified using two independent methods over the course of the experiments. One of these methods employed direct cell counting by taking 1 mL subsamples of culture suspension, fixing with 1% (v/v) paraformaldehyde, storing at 4°C before filtration (GTTP, 0.2 μm; Millipore), DAPI staining, and microscopic cell counts (Leica DM 5500 B epifluorescence microscope; Leica Microsystems, Wetzlar, Germany), as described previously (Rughöft et al., 2020 (link)). To compliment this method, functional marker genes were quantified as a measure of biomass, since in silico analysis using Geneious Prime (version 2019.1.1) and the NCBI data base (accessed in January 2019; Schoch et al., 2020 (link)) revealed that all investigated functional PAH-degrading genes were present as a single copy on the genome of Cycloclasticus pugetii strain PS-1 (Supplementary Table S1).