90i eclipse widefield microscope

Mycobacterial loads in lungs and spleen of Mtb-infected mice were determined at different time points post-infection as previously described (29 (link)). Lungs of Mtb-infected mice were fixed with 4% phosphate-buffered formalin, and 3 μm-thick sections were stained with either H&E or rabbit anti-mouse antibody specific for iNOS (Abcam) or rabbit anti-mouse IgA antibody (Abcam). Detection was performed using HRP-labelled anti-rabbit antibody (Dako) followed by 3, 3’-diaminobenzidine substrate (Dako). The lung images and lesion areas, iNOS and IgA positive areas were acquired in Nikon 90i Eclipse widefield microscope and quantified using NIS elements.

The immunized mice were challenged intratracheally on day 42 with a lethal dose of 3 × 10⁸ CFU, mixed with 10% porcine mucin (type 3; Sigma-Aldrich, Taiwan) of mid-log phase E. anophelis C08. At 36 hpi (when the control mice were expected to begin dying), organs were harvested and homogenized in sterile PBS. The homogenized organs were quantitatively cultured for each separate mouse to determine the bacterial burden. The excised organs were placed in vials containing 4% formaldehyde and stained with hematoxylin and eosin and myeloperoxidase antibody (ab188211, Abcam, Cambridge, UK) for immunohistochemical analysis of neutrophilic granulocytes. Multiple tissue sections of each organ were scored by a blinded pathologist for injury severity, as previously described (45 (link), 46 (link)). MPO-positive areas were imaged using a Nikon 90i Eclipse widefield microscope (Nikon Instruments Inc., Melville, NY, USA) and quantified using NIS-Elements software.

Mycobacterial loads in the lungs and spleen of Mtb-infected mice were determined as previously described (25 (link)). The right superior lobes were fixed with 10% neutral-buffered formalin, and tissue was processed with the Leica TP 1020 benchtop processor for 24 h and embedded in paraffin wax. Four 3 µm thick sections with 30 µm distance apart were cut in Leica Sliding Microtome 2000R, deparaffinized and subsequently stained with the hematoxylin & eosin (H&E) stain. The lung images were acquired in Nikon 90i Eclipse widefield microscope and free alveolar space was quantified using NIS elements (Nikon Corporation, Japan). Briefly, the images were converted to binary, and H&E positive area was measured. Fill holes function of NIS elements was employed to measure the complete lung area including the alveolar spaces. Free alveolar spaces were calculated by subtracting H&E positive area from the complete lung area and presented as a percentage to the complete lung area.