Online data supplement

Methods

Immunohistochemistry

Quantification of leukocyte and neutrophil homing after CPR was performed on 7 µm paraffin sections. The animals were perfused with 4% paraformaldehyde in phosphate buffer (pH 7.4). Organs were removed and postfixed in phosphate buffered 4% paraformaldehyde (pH 7.4) overnight prior to paraffin-embedding and sectioning. Sections were deparaffinized with xylene and dehydrated in graded series of ethanol. Endogenous peroxidase was quenched with 3% H₂0₂ in methanol for 30 minutes at RT. Sections were rinsed in 10 mM Tris, 150 mM NaCL, pH 7.4 (TBS) blocked with 20% swine serum in TBS for 45 min at RT. Neutrophils were detected with rat anti-neutrophils monoclonal antibodies, clone MCA771G (Serotec, Oxford, England), diluted 1:500 in TBS supplemented with 2% bovine serum albumin. Sections were incubated with primary antibodies overnight in a humidified chamber. Primary antibodies were detected with the avidin biotin complex technique using the ABC Elite Kit according to the manufacturer instructions (Vector Laboratories, Burlingame, CA, USA) and 3'-diamino benzidine tetrahydrochloride as chromogenic substrate. Sections were counterstained with Harris’ Hematoxylin and mounted in DPX (Serva, Heidelberg, Germany). Serial sections were photographed with a Zeiss Axiophot II (Zeiss, Oberkochen, Germany) equipped with a digital camera, colour view (SIS, Münster, Germany). 3 serial sections/mice were used to quantify neutrophils/high power field 24 h and 48 h after surgery. Homing of leukocytes and neutrophils was quantified using analySIS software (SIS, Münster, Germany).

Endothelial cells and ICAM-1 were stained using indirect immunofluorescent technique. Organs of Sham or CPR mice were snap-frozen in Tissue Freezing Medium (Leica Instruments, Nussloch, Germany). 10 µM cryosections were air dried overnight and fixed in cold acetone for 10 min. After blocking sections with PBS, supplemented with 1% bovine serum albumine and 0,05% Tween 20, endothelial cells were detected with polyclonal rabbit anti-von Willebrand Factor antisera (Dako, Glostrup, Denmark) diluted 1:200 in blocking buffer, ICAM-1 was stained with rabbit anti-ICAM-1 IgGs (Santa Cruz Biotechnology, Heidelberg, Germany) diluted 1:100 in blocking buffer for 1 h. Primary antibodies were visualized by incubating sections with Cy2-labeled goat anti-rabbit IgGs (Dianova, Hamburg, Germany), diluted 1:100 in blocking buffer for 1h. Sections were counterstained with DAPI Nucleic Acid Stain (Invitrogen, Carlsbad, CA, USA) and embedded in Fluorescent Mounting Medium (Dako, Glostrup, Denmark).

Western Blot

Snap frozen tissue samples were pulverized and homogenized (Micro-Dismembrator U and Homogenisator Modell 853202, B. Braun Melsungen, Melsungen, Germany) in 2fold concentrated reducing Laemmli gel loading buffer (2fold is: 120 mM Tris, 4% SDS, 20% glycerol, 0,05% bromphenol blue, 200 mM dithiothreitol, pH6,8) and separated by gel electrophoresis with 10% Tris/glycine SDS-polyacrylamide gels. The amount of protein per lane was adjusted by densitometric analysis (Kodak Image Station 1000 Kodak, New York, NY, USA) of coomassie brilliant blue stained was gels performed in parallel. Proteins were transferred from gel to PVDF-membranes (Millipore, Billerica, MA, USA) using the Mini Trans-Blot Cell (Biorad, Hercules, CA, USA). Membranes were blocked with 3% bovine serum albumin in PBS for 2h at RT and then incubated with polyclonal goat anti-ICAM-1 antibody (Santa Cruz Biotechnology, Heidelberg, Germany), diluted 1:200 in PBS supplemented with 1% bovine serum albumin. Membranes were washed with PBS with 0.1 % Tween 20 and incubated with horseradish peroxidase-labeled anti-goat IgGs (Santa Cruz Biotechnology, Heidelberg, Germany), diluted 1:4000 in PBS supplemented with 1% bovine serum albumin. Blots were developed with Super Signal West Pico chemiluminescent substrate (Pierce Biotechnology, Rockford, IL, USA) and the Kodak Image Station 1000.