Axiocam 503

For cryosections or paraffin embedding, testes were fixed for 4 hrs–overnight in either fresh 4% paraformaldehyde or Bouin’s solution, respectively, at 4°C and prepared as described previously [85 (link)]. Bouin’s-fixed testes were stained with Periodic Acid Schiff (PAS) using standard methods. For immunohistochemistry (IHC), immunostaining was performed on Bouin’s-fixed sections as described [85 (link)]. Brightfield images were captured on an Axio Observer A1 inverted microscope outfitted with a Zeiss Axiocam 503 color digital camera and Zen software (Carl Zeiss Microscopy, LLC).
For indirect immunofluorescence (IIF), immunostaining was performed on cryosections as described [85 (link)]. Alexa-Fluor conjugated secondary antibodies (Thermo Scientific) raised against the animal host of the primary antibody (Table 1) were incubated for 1 hr at room temperature at a 1:500 dilution. Coverslips were mounted for IIF with Vectastain containing DAPI (Vector Laboratories). Sections were imaged using a Fluoview FV1000 confocal laser scanning confocal microscope (Olympus America).

After harvesting the root nodules from the legumes, nodules were cut longitudinally with a razor blade and embedded in 4% agarose. After polymerization under vacuum conditions for 15min at room temperature, thin sections of 80–90μm were prepared with a Leica VT1000S Microtome. Sections were incubated in 100-fold diluted SYBR® green I nucleic acid stain (Sigma-Aldrich, Darmstadt, Germany) for at least 30min at 4°C. Morphology of the root nodules was investigated with an Olympus SZX12 stereo microscope equipped with Olympus DF PLAPO 1XPF objective lens (Olympus, Shinjuku, Tokio, Japan) coupled with a Zeiss Axiocam 503 color camera (Zeiss, Oberkochen, Germany).

Plasma membrane permeability was assessed by the passive uptake of propidium iodide (PI) (20 mM in dimethyl sulfoxide [DMSO]; Invitrogen). C. glabrata cell suspensions from strains KUE100 and KUE100_Δcgrpn4 were prepared in BM until a standard culture OD₆₀₀ of 0.5 was reached and transferred to the same medium with or without 150 mg/liter fluconazole. After 1 h of incubation, PI was added to 1 ml of 4 × 10⁷ cells/ml to a final concentration of 20 μM, and cell suspensions were incubated in the dark with orbital agitation (15 min at 250 rpm). Cells exposed to PI were centrifuged (17,500 × g for 5 min), washed twice, and resuspended in PBS for final aliquots of 10⁷ cells/ml. PI fluorescence was detected by fluorescence microscopy with a Zeiss Axioplan microscope (Carl Zeiss MicroImaging), using excitation and emission wavelengths of 536 and 595 nm, respectively. Fluorescence images were captured using a cooled Zeiss AxioCam 503 color camera (Carl Zeiss Microscopy), and the images were analyzed with ZEN lite software from Zeiss Microscopy. The cell-to-cell fluorescence intensity was defined as the average pixel-by-pixel intensity in the selected region of interest, and a minimum of 100 cells per experiment were used. The fluorescence images were background corrected by using dark-current images.

To evaluate morphological changes induced by the three different types of media, hAECs (7.5 × 10³ cells/well) and hDPSCs (5 × 10³ cells/well) were seeded on coverslips in 24-well plates and cultured as described above. After 7 days, cells were fixed with 4% formalin (10 min), permeabilized with 0.1% Triton X (5 min) and stained with phalloidin (30 min) and DAPI (1 min). Coverslips were mounted on slides (ProLong Glass Antifade Mountant, Thermo Fisher Scientific, Waltham, USA) and imaged on a ZEISS microscope (Axio Vert.A1, Carl Zeiss Microscopy, Jena, Germany) with the ZEISS Axiocam 503 color camera (Carl Zeiss Microscopy, Jena, Germany). Images with filters for blue (Carl Zeiss Microscopy, Jena, Germany) and red fluorescence (Set 43 and Set 49, Carl Zeiss Microscopy, Jena, Germany) in place were taken independently and digitally superimposed. ZEN software was used for microscopy and imaging (version 3.1, Carl Zeiss Microscopy, Jena, Germany).

24-hour cultures of C. albicans strains in YPD medium (28°C; 120 rpm) were centrifuged (5 min, 2260 x g). The collected cells were washed with fresh YPD medium and subsequently resuspended in YPD medium to OD₆₀₀ = 0.4. The exposure to AMF was performed in 8-well culture chambers, as described in “Exposure of C. albicans to AMF”. The hyphal transition was induced by cells suspension treatment with FBS (final conc. = 10%) for 2h at 28°C. After incubation, the samples were observed under a Zeiss Axio Imager A2 microscope equipped with a Zeiss Axiocam 503 mono microscope camera (Zeiss, Oberkochen, Germany) for the assessment of cell morphology (n = 50–100 cells in three repetitions). Zeiss ZEN 2 Blue software was used for the measurement of the length (μm) of straight hyphae.

Collected nodules were prepared as described in Reyero-Saavedra et al. (2017) (link). For pictures, safranine-stained semi-thin sections (25 μm) from twenty nodules of each strain detached 21 dpi were prepared using a hand-microtome and stained for 5 min with safranine in 50% ethanol before embedded in LR-White Resin were examined with a Zeiss AX10 microscope coupled to a Zeiss Axiocam 503 color digital camera (Carl Zeiss). Images were processed using ImageJ 1.52v.

A total of five photomicrographs from prepared jejunal tissue histology sections were taken at 10× magnification mounted on a glass slide and read under a Zeiss Axiocam 503 color camera (Carl Zeiss, Oberkochen, Germany) on a Zeiss Axioskop 40 microscope (Carl Zeiss, Oberkochen, Germany). Villus height (axis top to villous-crypt junction) and crypt depth (from villus-crypt junction to the base of villus) were measured with the ZEISS Efficient Navigation software (Carl Zeiss, Oberkochen, Germany). For the assessment, ten randomly selected well-oriented intact villus and crypts were measured per piglet and tissue to calculate mean villus height and crypt depth. Additionally, the villus to crypt ratio was calculated. All morphometric measurements were performed by the same researcher who was blinded to treatments and timepoints.