Axiocam 503 color

C. glabrata cell suspensions from strains KUE100, KUE100_∆cgmar1, and KUE100_∆cgrsb1 were prepared in BM until a standard culture OD600nm of 0.5 ± 0.05 was reached. C6 NBD-dihydrosphingosine (NBD-DHS; 1 mg/mL in methanol; Santa Cruz Biotechnology, Santa Cruz, CA, USA) was added to 1 mL of 4 × 10⁷ cells/mL to a final concentration of 5 µM, and cell suspensions were incubated in the dark with orbital agitation (30 min, 250 rpm). Cells exposed to C6 NBD-DHS were centrifuged (17,500× g for 5 min), washed twice, and resuspended in PBS buffer to final 10⁷ cells/mL aliquots. NBD fluorescence was detected by fluorescence microscopy in a Zeiss Axioplan microscope (Carl Zeiss MicroImaging, Oberkochen, Germany), using excitation and emission wavelengths of 395 and 509 nm, respectively. Fluorescence images were captured using a cooled Zeiss Axiocam 503 color (Carl Zeiss Microscopy, Oberkochen, Germany). Quantification of NBD-DHS signal localization to cell membranes was performed by accessing a minimum of 100 cells per strain.

Plasma membrane permeability was assessed by the passive uptake of propidium iodide (PI; 20 mM in DMSO, Invitrogen, Waltham, MA, USA) as described previously [26 (link),29 (link)]. C. glabrata cell suspensions from strains KUE100, KUE100_∆cgmar1, and KUE100_∆cgrsb1 were prepared in BM until a standard culture OD600nm of 0.5 ± 0.05 was reached and transferred to the same medium with or without 150 mg/L of 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, 250 rpm). Cells exposed to PI were centrifuged (17,500× g for 5 min), washed twice, resuspended in PBS buffer, and pooled to final 10⁷ cells/mL aliquots. PI fluorescence was detected by fluorescence microscopy in 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 (Carl Zeiss Microscopy), and the images were analyzed with the ZEN lite software from ZEISS microscopy. Cell-to-cell fluorescence intensity was defined as the average of pixel-by-pixel intensity in the selected region of interest, and a minimum of 70 cells per strain were assessed. The fluorescence images were background corrected using dark-current images.

For histologic evaluation, enteroids from one well of a 24 well plate were harvested and fixed with 2% v/v formaldehyde (ROTI^® Histofix 4%, Carl Roth GmbH + Co. KG, Karlsruhe, Germany) for 20 min at room temperature. Native jejunum and colon samples of the same horses were fixed with 4% v/v formaldehyde for 24 h and stored in 70% ethanol at 4 °C. Formalin-fixed cryosections (6 µm) of enteroids and the corresponding tissue samples were stained using haematoxylin and eosin (H&E) as well as Periodic acid-Schiff (PAS).
Additionally, immunohistochemical (IHC) stainings of chromogranin A (CHGA) as a marker for enteroendocrine cells were performed with paraffin-embedded sections (2.5 µm). Therefore, the samples were blocked with 1.5% goat serum (Merck, Vienna, Austria) diluted in PBS, incubated over night at 4 °C with primary antibody (Anti-Chromogranin A, rabbit polyclonal IgG antibody, Abcam, Cambridge, UK; #ab45179; 1:20 000), for 30 min with secondary antibody (Goat Anti-Rabbit HRP, Medac GmbH, Wedel, Germany; #DPVR110HRP) according to the manufacturer’s instructions and counterstained with haematoxylin acidic. Pictures were taken with a Zeiss Axiocam 503 color (Carl Zeiss Microscopy GmbH, Oberkochen, Germany).

Formalin‐fixed and paraffin‐embedded (FFPE) tissue samples of human colon cancer moderately differentiated (G2) cases were selected for in situ immunophenotypic analyses. Four‐micrometers‐thick sections were deparaffinized, rehydrated, and unmasked using Novocastra Epitope Retrieval Solutions pH 9 in a thermostatic bath at 98 °C for 30 min. Subsequently, the sections were brought to room temperature and washed in PBS. After neutralization of the endogenous peroxidase with 3% H₂O₂ and Fc blocking by a specific protein block (Leica Novocastra, Wetzlar, Germany), the samples were incubated with phospho‐DRP1 (Ser 616) (clone D9A1 Cell Signaling, 1 : 100) and PD‐1 (clone NAT105 Abcam, 1 : 50) antibodies. IHC staining was revealed using MACH 2 Double Stain 1 kit (Biocare, Pacheco, CA, USA) and DAB (3,3'‐diaminobenzidine, Leica Novocastra) and Vulcan Fast Red as substrate chromogens. Triple immunostainings were performed by incubating the same sections with CD8 antibody (clone 4B11, 1 : 50 pH9, Leica Novocastra) and anti‐mouse Alexa Fluor 488‐conjugated secondary antibody (1 : 500, Life Technologies, Carlsbad, CA, USA). Slides were analyzed under a Zeiss Axioscope A1, and microphotographs were collected using a Zeiss Axiocam 503 Color with the Zen 2.0 Software (Zeiss).

The presence of intracellular ROS was assessed by the cell-permeant 2ʹ,7ʹ-dichlorodihydrofluorescein diacetate indicator (H₂DCFDA; 25 mg/mL in DMSO, Santa Cruz Biotechnology, Inc.). C. glabrata cell suspensions from strains KUE100 and KUE100_∆cgtog1 were prepared in BM until a standard culture OD_600nm = 0.5 ± 0.05 was reached and transferred to the same medium with or without 15 mM H₂O₂. After 1 h of incubation, cells were centrifuged (17,500 × g for 5 minutes), washed twice and resuspended in PBS buffer to a final 10⁷ cells/mL aliquots. H₂DCFDA was added to 1 mL of 4 × 10⁷ cells/mL to a final concentration of 0.2 µL/mL and cell suspensions were incubated in the dark with orbital agitation (20 minutes, 250 rpm). Cells exposed to H₂DCFDA were centrifuged (17,500 × g for 5 minutes), washed twice and resuspended in 1 mL PBS buffer. H₂DCFDA-fluorescence was detected by fluorescence microscopy in a Zeiss Axioplan microscope (Carl Zeiss MicroImaging), using excitation and emission wavelength of 395 and 509 nm, respectively. Fluorescence images were captured using a cooled Zeiss Axiocam 503 color (Carl Zeiss Microscopy) and the images were analyzed with the ZEN lite software from ZEISS microscopy. Cell-to-cell fluorescence intensity was defined as the average of pixel by pixel intensity in the selected region of interest and a minimum of 100 cells/experiment were used.

Vertebral bodies no. 32–35 and 36–39 were dissected for serial sectioning of previously non-demineralized or demineralized samples (two samples/group) (Figure 1). For embedding in glycol metacrylate (GMA), samples were treated as described in [38 (link)]. Sections were stained with toluidine blue, a metachromatic stain which visualizes skeletal and cartilaginous tissues, cellular structures, and collagen fibers. von Kossa/Van Gieson was used to visualize phosphate in the mineralized bone and type I collagen bone matrix [72 ]. Sections were cover slipped with DPX (Sigma-Aldrich, Overijse, Belgium) and observed using a Zeiss Axio Imager-Z compound microscope (Carl Zeiss Microscopy GmbH, Jena, Germany) equipped with an Axiocam 503 color camera.