Goat anti chicken alexa fluor 594

Immunofluorescence analysis of HBMEC grown in chamber slides and HIBCPP cells cultivated in the inverted cell culture insert model was performed as previously described [29 (link)]. The following antibodies were used: primary antibodies: chicken anti-vimentin (BioLegend, San Diego, CA, USA), goat anti-Met (Abcam, Cambridge, UK), mouse anti-Ecad (BD, Franklin Lakes, NJ, USA), and rabbit anti-ZO-1 (Invitrogen, Carlsbad, CA, USA); secondary antibodies: goat anti-chicken Alexa Fluor^® 594, donkey anti-goat Alexa Fluor^® 594, goat anti-mouse Alexa Fluor^® 594, chicken anti-rabbit Alexa Fluor^® 488, and donkey anti-rabbit Alexa Fluor^® 488 (all Invitrogen, Carlsbad, CA, USA). Nuclei were stained with 4′-6-diamidino-2-phenylindole dihydrochloride (DAPI) (1:50,000 in PBS/1% BSA) (Merck, Darmstadt, Germany). Densitometric analysis of Western blot bands normalized to actin was performed using the ImageJ software 1.53e [48 (link)].

CG4+ and CG8+ cartridges were purchased from Abbott (Princeton, NJ, USA). Chicken anti-C3/C3a, mouse anti-C4d, mouse anti-C5b-9, and mouse anti-endothelial cell antibodies were obtained from Abcam Inc. (Cambridge, MA, USA). Rabbit anti-C5 antibody was from Abbiotec, LLC (San Diego, CA, USA). Mouse anti-IL-6 was purchased from R&D Systems (Minneapolis, MN, USA). Mouse anti-porcine C3a, biotinylated anti-C3a, and porcine C3a standard were from Georg-August-Universität Göttingen Stiftung Öffentlichen Rechts (Göttingen, Germany). Goat anti-chicken Alexa Fluor 594, goat anti-mouse Alexa Fluor 488, goat anti-mouse Alexa Fluor 594 IgG (H+L) conjugated secondary antibodies, and ProLong Gold antifade reagent were from Invitrogen (Carlsbad, CA, USA).

Confluent HUVEC were grown on glass coverslips, some of which were treated for 24 h with CITCO (3 µM) or vehicle, and then stimulated for 24 h with TNFα (20 ng/mL). The cells were then washed with PBS, fixed with 4% paraformaldehyde and blocked in PBS/1% BSA solution. Protein localization was detected by indirect immunofluorescence using the following primary antibodies: rabbit monoclonal anti-human CAR (1:50 dilution, cat# ab186869, Abcam, Waltham, MA, USA), mouse monoclonal anti-human VCAM-1 (1:50 dilution, cat# MCA907, BioRad, Barcelona, Spain), mouse monoclonal anti-human phosphorylated-NFκB (1:100 dilution, cat# 610869, BD Biosciences, Madrid, Spain) and goat polyclonal anti-human RXRα (1:50 dilution, cat#ab24636, Abcam, Waltham, MA, USA). Immunofluorescence signals were detected using the following secondary antibodies: Alexa Fluor 488 goat anti-mouse (1:1000 dilution, cat# A11001), Alexa Fluor 488 goat anti-rabbit (1:1000 dilution, cat# A11034) or Alexa Fluor 594 chicken anti-goat (1:1000 dilution, cat#A21468), all from Molecular Probes (Life Technology, Eugene, OR, USA). To confirm specificity of antibodies, isotype controls (cat# 172730; cat# ab18451, respectively, both from Abcam, Waltham, MA, USA) or secondary antibodies only were used as negative controls. Nuclei were stained with 4′,6-diamidino 2-phenylindole (DAPI, Thermo Fisher Scientific, Waltham, MA, USA).

AT samples were fixed and mounted in paraffin and sectioned into 5 μm slides with a microtome (Leica Biosystems, Nussloch, Germany). Antigen was unmasked with proteinase K (cat#S3020, Dako, Santa Clara, CA) and blocked with 15% horse serum for 1 h. Samples were incubated with the following primary antibodies overnight at 4°C: mouse anti-human CCL17 (1:50, cat#DY364-05, R&D Systems), mouse anti-human CCL22 (1:50, cat#DY336, R&D Systems), goat anti-human CCR4 (1:100, ab1669; Abcam, Cambridge, UK), rabbit anti-human CD3 (1:100, cat#C7930, Sigma-Aldrich, St. Louis, MO), rabbit polyclonal anti-human CD31 (1:50, cat#ab32457, Abcam) and rat anti-human Mac-3 (1:100, cat#sc19991, Santa Cruz Biotechnology, Dallas, TX). Alexa-Fluor^® 488 goat anti-rabbit (cat#A11034), Alexa-Fluor^® 488 donkey anti-rat (cat#A21208), Alexa-Fluor^® 594 goat anti-rat (cat#A1107), Alexa-Fluor^® 594 goat anti-mouse (cat#A1105) and Alexa-Fluor^® 594 chicken anti-goat (cat#A21468) antibodies were used as secondary antibodies (dilution 1:1000; all from Molecular Probes, Eugene, OR). Nuclei were stained with Hoechst (1:4000). Afterwards, five fields from each section were captured with a Zeiss Axio Observer A1 fluorescence microscope (Carl Zeiss Micro Imaging GmbH, Oberkochen, Germany).

Sections were washed in 3 × 5-min changes of PBS and blocked for 20 min in 5% PBS/goat serum (Sigma-Aldrich, Dorset, UK). Sections were incubated at 4°C overnight with chicken anti-GFAP (1:100) (Cat no. abs4674) (Abcam, Cambridge, UK). Sections were washed 3 × 5 min in PBS and incubated at room temperature for 1 h with Alexa Fluor 594 goat anti-chicken (1 in 500 in PBS) (ThermoFisher, UK). Sections were washed 3 × 5 min and mounted using Vectashield mounting medium with DAPI (Vector Labs, Peterborough, UK).