Ab188211

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.

Five µm cross-sections of the abdominal aorta and the aneurysm were used for detection of elastin by Miller’s elastin staining (Atom Scientific, Hyde, UK) according to the manufacturer’s instructions. Other aneurysm cross-sections were used for detection of neutrophils (myeloperoxidase, MPO, 1:8000, ab188211, Abcam, Cambridge, UK) and macrophage marker Cluster of Differentiation 68 (CD68, 1:500, ab125212, Abcam, Cambridge, UK) using the protocol previously described by Melin et al. [30 (link)]. For both labeling protocols, rabbit immunoglobulin (X0903, DAKO, Agilent, Glostrup, Denmark) in the corresponding concentration was used as a negative control.
Micrographs were captured using an Olympus Bx51 microscope with an attached Olympus DP26 camera. ImageJ software (ImageJ 1.53a Wayne Rasband, National Institutes of Health, Bethesda, MD, USA) was used to quantify histological stainings and identify tunica media. The percentage of elastin remaining in tunica media was quantified with a color threshold tool. In addition, the degree of elastin degradation in tunica media in Miller’s stained sections were divided into 8 areas and scored from 1–4, 1 being organized elastin with no ruptures and 4 being the total disruption of complete concentric elastin lamellae by 2 investigators. The individual variation was 5.67%; thus, the average score was used for analysis.