U cmad3 camera

Before excising lungs, number of visual lung lesions in all lobes was counted in M.tb-infected mice. Lungs were fixed in Excell plus fixative (American MasterTech Scientific Inc.) for at least 48 h before processing them for paraffin embedding. Thin sections (5 µm) were stained with hematoxylin and eosin (H&E) for histopathological examination. Immunohistochemistry was performed essentially as described in an earlier publication [38] (link). H&E and immune-stained sections were viewed under Olympus BX50 microscope and photographed with Olympus UCMAD3 camera using Picture Frame software. The same exposure settings were used for all image capturing. For determination of granuloma size, images captured at 4× magnification were used to measure granuloma diameter using Nikon NIS-Elements BR 3.2 software. TF expression levels in the granuloma was estimated based on the intensity of the immunopositive areas by scoring semi-quantitatively as absent, slight, moderate or strong with assigned scores of 0, 1, 2 and 3, respectively. Total 10–20 granulomas were analyzed to calculate either average granuloma size or scoring TF staining intensity by two investigators.

Aqueous dispersions of Kraft birchwood cellulosic fibers (kindly provided by Sandra Tapin, FCBA, Grenoble, France) were adjusted to pH 5.2 with acetate buffer (50 mM) in a final reaction volume of 5 ml. Each GcLPMO9 enzyme was added to the fibers at a final concentration of 20 mg g⁻¹ in the presence of 1 mM of ascorbic acid. Enzymatic incubation was performed at 40 °C under mild agitation for 48 h. Samples were then dispersed using a Polytron PT 2100 homogenizer (Kinematica AG, Luzern, Switzerland) for 3 min, and ultrasonicated by means of a QSonica Q700 sonicator (20 kHz, QSonica LLC., Newtown, USA) at 350-W ultrasound power for 3 min as previously described [20 (link)]. The reference sample was submitted to the same treatment but did not contain the enzyme. Wood cellulose fibers (reference and GcLPMO9-treated) were deposited onto a glass slide and observed under a BX51 polarizing microscope (Olympus France S.A.S.) with a 4 × objective. Images were captured on a U-CMAD3 camera (Olympus, Tokyo, Japan).

The non-fluorescent co-aggregation assay was based on^{26 (link)} with several adjustments. Overnight C. albicans SC5314 cultures were washed twice (1500 × g, 10 min) and resuspended in phosphate buffered saline (PBS) to obtain a 1% (w/v) cell suspension. S. cerevisiae CNCM I-3856 from commercial dried powder was also resuspended in PBS to obtain a 1% w/v cell suspension. Lactobacillaceae cultured overnight were adjusted to a concentration of 10¹⁰ CFU/mL. After vortexing, 50 µL of C. albicans suspension, 50 µL of S. cerevisiae suspension and 25 µL of lactobacilli were added to the wells of a U-bottom 96-well plate (VWR). All Lactobacillaceae and S. cerevisiae were also tested as such with C. albicans. The plate was incubated at room temperature with gentle shaking. After 10 min and 1 h, the co-aggregation rate was microscopically evaluated using the Olympus CX41 light microscope and Olympus U-CMAD3 camera. Scores from 0 to 4 were given for each condition, as described by^{26 (link)}, with a score of 0: no aggregation; 1: aggregates with small clusters; 2: aggregates with larger numbers of yeasts; 3: clumps visible with the naked eye containing large numbers of yeast cells; 4: maximum score for large clumps visible with the naked eye in the well center. For conditions where even higher aggregation was observed, we rationalized that a higher score was needed and indicated this with 4+.

Tenocytes were plated at a density of 50 000 cells/ml on coverglasses in 24-well plates and allowed to adhere overnight. Media was then changed to either high or low glucose with or without peroxide for a further 8 h. Cells were then fixed with 10% formaldehyde for 30 min. Following washing, cells were permeabilised with 0.5% triton-X in PBS for 5 min. Non-specific binding sites were blocked by incubating in 3% horse serum for 1 h at room temperature before incubation in FOXO1 primary antibody (diluted 1 : 50) for 18 h at 4 °C in a humidified chamber. Following thorough washing, cells were incubated with Dylight-488 anti-rabbit secondary antibody (Thermo-Fisher Scientific Ltd, Rockford, IL, USA; diluted 1 : 100 with PBS) for 45 min at room temperature, and then 4′6-diamidino-2-phenylindole dichloride (DAPI) for a further 30 min at room temperature. Cells were then thoroughly washed with ddH₂O before mounting in VectaShield (Vector Laboratories Inc, Burlingame, CA, USA) and imaging on an Olympus BX40 microscope using an Olympus U-CMAD3 camera (Olympus, Southend-on-Sea, UK). IgG and secondary antibody controls were used to verify the specificity of the primary antibody.