Axiocam 506 mono camera

To estimate the abundance of Ca. Nitricoxidivorans perseverans and Ca. Nitricoxidireducens bremensis in the enrichment culture and the denitrifying sludge from which it was inoculated, CARD–FISH was performed on filter pieces with cells from the enrichment culture and the inoculum using Nper205 and Nbre448 probes as described elsewhere^{91 (link)}. Nper205 and Nbre448 probes required formamide concentrations in the hybridization buffer of 40% and 30% (v/v), respectively. Positive and negative controls with an equimolar mixture of probes EUB338-I, EUB338-II and EUB338-III^{92 (link),93 (link)}, and with probe NON338 (ref. ^{94 (link)}) were included. Following CARD–FISH, cells were stained using DAPI to target DNA of all microorganisms. Relative abundances of Ca. Nitricoxidivorans perseverans and Ca. Nitricoxidireducens bremensis were determined from CARD–FISH and DAPI counts (n ≥ 1,000) in triplicate filter pieces that were either hybridized with Nper205 or with Nbre488. Double CARD–FISH was performed to simultaneously visualize cells of Ca. Nitricoxidivorans perseverans and Ca. Nitricoxidireducens bremensis by hybridization with probe Nper205 followed by inactivation of peroxidases and a second hybridization step with probe Nbre448. The Zeiss Axio Imager M2 epifluorescence microscope equipped with an Axiocam 506 mono camera (Zeiss) was used for cell counting and image acquisition.

Slides were viewed using a ZEISS AxioImager M2 automated microscope with Axiocam 506 mono camera (ZEISS, Gottingen, Germany). An A-Plan 20 ×/0.45 numerical aperture Ph2 objective was used to capture images for the quantification of fibre type distribution and capillarisation. A Plan-Apochromat 40 ×/1.3 numerical aperture oil Ph3 [UV] VIS-IR objective (Gottingen, Germany) was used with Apotome.2 structured illumination to capture detailed images of myoglobin and mitochondrial content. Identical camera settings were used to capture all images between participants on the same glass slide. The 4′-6-diamidino-2-phenylindole (DAPI) UV (340–380 nm) excitation filter was used to visualise Alexa Fluor 350 fluorophores (blue; myosin heavy chain I within type I muscle fibres), the FITC (465–495 nm) excitation filter was used to visualise Alexa Fluor 488 fluorophores (green; dystrophin and capillaries), and the Texas-Red (540–580 nm) excitation filter was used to visualise Alexa Fluor 594 fluorophores (myoglobin and COXIV as a marker of the mitochondria). For the duplicate cross-sections, 10 images were captured per cross-section (totalling 20 per participant).

Cells were cultivated on cover slips, washed with PBS, and fixed for 20 min with 4% (wt/vol) PFA (P6148; Sigma-Aldrich) in PBS. After washing with PBS, they were permeabilised for 5 min with 0.5% (vol/vol) Triton X-100 in PBS and washed twice with PBS. Epitopes were blocked for 1 h with 2% (wt/vol) BSA and 0.05% (vol/vol) Triton X-100 in PBS. Then, the cells were incubated for 1 h with the primary antibodies diluted in the blocking solution, washed thrice for 5 min with PBS, and incubated for 1 h with the secondary antibodies and Hoechst 33342 (1:3,000) diluted in 0.05% (vol/vol) Triton X-100 in PBS. After washing thrice for 10 min with PBS, they were post-fixed for 5 min with 1% (wt/vol) PFA in PBS, washed with PBS, and mounted with ProLong Gold Antifade Mountant (P10144; Thermo Fisher Scientific). Photomicrographs were taken with an Axiovert microscope (Carl Zeiss) equipped with a Axiocam 506 mono camera (Carl Zeiss) at 10× and 20× magnification or with a confocal microscope (Leica SP8 from Leica) at 63× magnification.