Ab37373

All experimental setups were permitted by the Bavarian Animal Care and Use Committee (ethical approval code: ROB-55.2Vet-2532.Vet_02-17-99, approved on 8 December 2017) and were conducted in strict accordance with the German animal legislation guidelines. Mice were fed a standard laboratory diet and were housed in a temperature-controlled room on a 12 h light–dark cycle. For all experiments, adult male SV-129 (Charles River Laboratories, Sulzfeld, Germany) mice, aged 8–12 weeks, were sacrificed at 24 h or 7 days (n = 5 per group) after the surgical procedure. 30 minutes prior to surgical intervention, mice were either treated i.v. with a neutralizing anti-CIRP antibody (Abcam, ab106230, Cambridge, UK, 1 mg/kg), an isotype antibody (Abcam, ab37373, 1 mg/kg), or phosphate-buffered saline (PBS, PAN Biotech, Aidenbach, Germany, pH 7.4, 1 mL/kg) and then two, four and six days after surgery. To ascertain the proliferation rate of endothelial cells in the lower hindlimb 7 days after surgery, SV-129 mice were daily injected with 100 µL BrdU (bromodeoxyuridine) (Sigma-Aldrich, St. Louis, MO, USA) (12.5 mg/mL BrdU in PBS) i.p., starting directly after the surgical intervention.

For inhibition experiments, representative lymphoma cell lines were pre-incubated with anti-ITGB1 (abx011001, Abbexa, Cambridge, UK), anti-ITGB2 (ab131044, Abcam, Cambridge, UK), anti-ITGB7 (Santa Cruz Biotechnology, Dallas, TX, USA), or anti-CDH2 (HPA046119, Sigma-Aldrich) antibodies for 30 min at 4 °C. The dilution of antibodies was 1:100. Cells treated with goat IgG (ab37373, Abcam) served as control. Next, the cells were centrifuged at 1800 rpm for 10 min at 4 °C, washed twice in PBS, and resuspended in fresh RMPI medium prior to evaluation of adhesion to mesenchymal stromal cells in time-scale in optical tweezers.
The percentage of lymphoma cells that stable bond to stromal cells within 40 and 300 s in optical tweezers were calculated for Ri-1 and U2904 cell line, respectively. Data were expressed as mean  ±  SEM in tree independent experiments for 30 cells for each experimental condition.

The cutting of sections and antigen retrieval was finished as described above. The sections were first incubated by goat anti-human Perilipin A antibodies (Ab61682, Abcam) with the co-staining of mouse anti-human GFP antibodies (66002-1-Ig, CMC) for 24 hours, then incubated by the second antibodies i.e. Alexa Fluor^® 555 donkey anti-goat IgG antibodies (A-21432, Invitrogen) or Alexa Fluor® 488 donkey anti-mouse IgG antibodies (A-21202, Invitrogen), counter-stained with DAPI (D-21490, Invitrogen). The isotype control group included goat polyclonal IgG (ab37373, Abcam) and mouse IgG1 (ab18448, Abcam). Fluorescent images were observed with fluorescence microscope, and taken photos of by digital microscope camera.

Immunoprecipitation reactions were as described in [18 (link)]. HEK 293T cells were allowed to reach confluence in 15 cm petri dishes (n = 4) and harvested in polysome lysis buffer (PLB) (10 mM Hepes, pH 7.0, 100 mM KCl, 5 mM MgCl₂, 0.5% NP-40, 1 mM DTT, 100 units/ml RNase OUT and protease inhibitors). Each plate yielded approximately 200 μl of lysate, 100 μl of which was used in each DJ-1 and IgG control IP. DJ-1 (Abcam, ab4150) or IgG isotype control (Abcam, ab37373) antibodies were conjugated to Protein G Dynabeads (Invitrogen) for 10 min at RT with rotation. The conjugated beads were incubated with cell lysate in NET2 buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1 mM MgCl₂, 0.05% NP-40, 20 mM EDTA, 1 mM DTT, 100 units/ml RNase OUT) for 1 h at RT and then washed six times with cold NT2 buffer.

For analyzing MIF receptor expression via confocal fluorescence microscopy, 300.000 freshly isolated cardiomyocytes were previously counted with a hemocytometer and subsequently seeded into μ-dishes (35 mm high, Ibidi, Munich, Germany) and cultivated for 12 days. Cells were then fixed with 3.6% paraformaldehyde (supplemented with 0.1% Hoechst 33342 for nuclear staining) for 20 min and subsequently permeabilized with 0.3% Triton X-100 and 5% FCS in PBS for 1 h. Antibodies against murine CD74 (C16, sc-5438, Santa Cruz Biotechnology, Heidelberg, Germany), CXCR2 (bs1629R, Bioss, Woburn, USA), and CXCR4 (ab2074, Abcam, Cambridge, USA) or appropriate isotype controls goat IgG (ab37373, Abcam, Cambridge, USA) or rabbit IgG (sc-2027, Santa Cruz Biotechnology, Heidelberg, Germany) were diluted 1∶200 in PBS (containing 0.3% Triton X-100 and 2% BSA) and cardiomyocytes were incubated for 2 h at room temperature. Following 3 washing steps with PBS fluorescently labeled secondary antibodies (Alexa Fluor 633 goat anti-rabbit IgG, Alexa Fluor 633 donkey anti-goat IgG Invitrogen, Darmstadt, Germany) were also diluted 1∶200 in PBS (containing 0.3% Triton X-100 and 2% BSA) and incubated with the cardiomyocytes for 1 h at room temperature. Stained cardiomyocytes were analyzed by laser scanning fluorescence microscopy using an LSM710 confocal microscope (Zeiss, Jena, Germany).