Ab22506

Animals were sacrificed by i.v. injection of potassium chloride (B. Braun Medical Oy, Helsinki, Finland) immediately after completing the PET/CT scan and the coronary arteries were prepared. Samples of the proximal LAD, proximal LCX and proximal RCA coronary arteries of approximately 3 to 5 cm long together with a blood sample were collected. Samples were weighed and radioactivity was measured using the gamma counter (1480 Wizard 3″; PerkinElmer/ Wallac, Turku, Finland). The radioactivity concentration was expressed as SUV = ([organ activity/organ weight]/[total given radioactivity/animal body weight]). In addition, vessel-to-blood ratios were calculated.
The coronary artery samples were mounted, frozen in isopentane mixed with dry ice, and cut longitudinally into serial 7 μm and 40 μm cryosections. Samples of 40 μm were immediately exposed overnight against a phosphor imaging plate (BAS-TR2025, Fuji Photo Film Co. Ltd., Tokyo, Japan). The distribution of radioactivity on the plate was visualized and quantified using Fluorescent Image Analyzer (Fujifilm FLA-5100, Fuji Photo Film Co. Ltd., Tokyo, Japan). The 40 μm cryosections were stained with hematoxylin & eosin (HE). The sections of 7 μm were stored at -70°C and stained with HE, Movat's pentachrome and macrophage staining (MAC387, ab22506, Abcam, Cambridge, MA, USA).

Tissue sections were also used to localize calprotectin using mouse antibody ab22506 (1:5000; Abcam) recognizing S100A9, followed by goat anti mouse IgG-Alexa 488 (1:100), based on a protocol described previously [38 (link)]. The calprotectin antibody has been used previously in equine tissues [16 (link), 45 (link), 46 (link)]. To confirm identity of keratinocytes, mouse monoclonal anti-keratin 14 antibody (Novus Biologicals; LL02, 1:500) was used in place of the anti-calprotectin antibody [47 ]. Rhodamine-tagged wheat germ agglutinin (12.5 μg/ml; Vector Laboratories) was used as a counterstain to outline cell membranes and extracellular matrix, facilitating identification of lamellar microanatomy [37 (link)]. Sections were imaged using a Zeiss 880 confocal microscope system including a Zeiss Axio Observer inverted microscope stand and 25X PlanApo objective (0.8 NA), controlled by Zen software (version 2.1). Large areas of tissue were imaged using the tile scan feature, typically set to 0% overlap between sections. The tiled sections were stitched together using Zen software (version 2.1).

Tissue sections were stained with hematoxylin and eosin (HE) using standard protocols. TUNEL staining was performed with an ApopTag kit (Millipore, Billerica, MA, USA) based on the manufacturer’s instructions. The antibodies for immunohistochemistry used in this study were those against Kim-1 (ab78494, Abcam), 4-hydroxynonenal (4-HNE, ab46545, Abcam), neutrophil gelatinase-associated lipocalin (NGAL, ab63929, Abcam), Ly-6B (NBP2-13077), NFκB p65 (sc-372, Santa Cruz, CA, USA), and MAC387 (ab22506, Abcam). Sections were treated with the Envision+ DAB kit (Dako, Basel, Switzerland) according to the manufacturer’s instructions.

Isolated leukocyte and lymphocyte samples were fixed in 4% paraformaldahyde in PBS (0.2 mM and pH 7.4) and spun in a cytocentrifuge (8 min at 300g) onto coated slides. Slides were permeabilized and blocked with 10% normal donkey serum in PBS (with 0.5% Triton X-100), and primary antibodies (S100A8/9, Abcam, ab22506; IFITM1, Abcam, ab233545) were incubated at 4 °C overnight, followed by incubation with donkey anti-rabbit Alexa Fluor 488 (Invitrogen, A32790) or anti-mouse Alexa Fluor 555 (Invitrogen, A31570) for 1 h at room temperature. Slides were sequentially stained with CD24 (Abcam, ab202073) on the same slides for 1 h at room temperature, followed by donkey anti-rabbit Alexa Fluor 647 (Invitrogen, A31573). Imaging was done using a VS-120 slide scanner (Olympus), and high-resolution imaging was done using an SP8 spectral confocal microscope (Leica). Image processing was completed in Fiji (version 2.1.0)^{60 (link)}.

Mouse left lungs were inflated with 1% low melt agarose (SLBH4354V; sigma, USA) at a pressure of 25 cm H₂O and then fixed with 4% neutral paraformaldehyde for 24 hours. Tissues were embedded in paraffin, sectioned (5 μm) and stained with haematoxylin and eosin (H&E). Mean linear intercept (Lm) was measured to show this verity of alveolar destruction as we previously described.14 Briefly, Lm was calculated in each sample based on 10 random fields and observed at a magnification of ×200 using a cross‐line. The sample slides were incubated with primary antibody against Mac‐3 (ab22506; Abcam, USA,1:1000) and Ly6G (sc‐168490; Santa Cruz, 1:250) for detecting of lung macrophages and neutrophils by IHC, respectively. After staining, the slides were examined under a light microscope by a photograph documentation facility (Olympus, Tokyo, Japan). Integrated optical density (IOD) of positive cells in each sample (at least 3 random microscopic fields per lung section, ×400) was analysed by Image pro‐plus 6.0 software.15