Goat anti rabbit igg cy5

The firefly luciferase reporter plasmid for IFN-γ-responsive promoter and the Renilla luciferase control plasmid (pRL-TK) (31 (link)–33 (link)) were kindly provided by Dr. Hong-Bing Shu (Wuhan University, China). The NSs or NP expression plasmids were constructed as described previously (10 (link), 13 (link), 18 (link)). Mouse anti-NSs antiserum or rabbit antisera to NSs, NP, GP (N-terminal domain), or RdRp were respectively raised against the corresponding viral proteins generated by Escherichia coli (10 (link), 18 (link)). Rabbit antibodies to S-tag (Abcam), STAT1 (Cell Signaling Technology), or STAT2 (Santa Cruz Biotech) and mouse antibodies to HA-tag (Beyotime), β-actin (Beyotime), or STAT1 (Santa Cruz Biotech) were purchased from the indicated manufacturers. Secondary antibodies include goat anti-mouse IgG-fluorescein isothiocyanate (FITC) (Proteintech), goat anti-rabbit IgG-Rhodamine (Chemicon), and goat anti-rabbit IgG-Cy5 (Abcam). Recombinant mouse or human IFN-γ proteins were purchased from Cell Sciences or Peprotech Inc., respectively. Recombinant human IFN-α was from PBL Biomedical Laboratories.

Screening of a UniPEx1 protein macroarray (BioScience ImaGenes) was performed according to the manufacturer’s protocol with purified recombinant antibody, either dAb or dAb-rFc. Briefly, the activated membrane was blocked with 5% BSA for 1 h before incubation overnight with 250 μg dAb or 125 μg dAb-rFc. Detection was performed by incubation for 2 h with secondary antibody; Monoclonal Anti-c-Myc − Cy3™ (Sigma-Aldrich) 1:2000 or Goat anti-Rabbit-IgG-Cy5 (Abcam, ab6564) 1:8000. The membrane was scanned with a Typhoon TRIO variable mode imager (Amersham Biosciences). Antibody binding to the proteins on the array was evaluated using an in-house developed Matlab program. The immobilised proteins were situated on the membrane in defined duplicated patterns arranged in squares consisting of 3x3 spots with a central ink dot. By evaluating the intensities of different spots compared to the background, as well as their positions, the program created a list of possible antigen hits listed from highest to lowest intensity. Only hits detected with both dAb and dAb-rFc formats were considered. Furthermore, hits detected only once despite being spotted several times in duplex were omitted.

Dexamethasone was from Sigma. Helenalin and SC-514 were from Merck Biochemicals. SB203580, PD98059, and LY294002 were from Enzo Life Sciences. Goat anti-human TNFα was from PeproTech. Mouse anti-human MMP-9, rabbit anti-M.tb, and goat anti-rabbit IgG Cy5 were from Abcam. Rabbit anti-human phospho-Akt, total-Akt, phospho-p38, total-p38, phospho-ERK, total-ERK, phospho-JNK, total-JNK, and goat anti-rabbit HRP linked were from Cell Signaling Technology. Rabbit anti-human neutrophil elastase was from Dako, and Mouse anti-human MMP-9 was from Millipore.

The broad spectrum MMP inhibitor GM6001 was from Calbiochem (Hertfordshire, UK). FITC-conjugated anti-integrin α2 (clone AK7, Abcam, Cambridge, UK), anti-integrin α3 (clone 17C6, Abcam), and IgG1 isotype control antibodies (BD Biosciences, Oxford, UK) were used in FACS assays. Anti-integrin α2β1 (clone BHA2.1) and anti-integrin α3β1 (clone M-KID2) antibodies were from Millipore (Hertfordshire, UK). Rabbit anti-MMP-1 primary antibody (Millipore) and Cy5-goat anti-rabbit IgG (Abcam) secondary antibody were used in confocal assays. Phalloidin conjugated with Alexa Fluor 594 (Thermo Fisher Scientific, Paisley, UK) was used for F-actin staining and DAPI was used as nuclear counterstain. Other reagents were purchased from Sigma-Aldrich (Dorset, UK) unless otherwise stated.

Slices were rinsed in 0.3% Triton X-100/PBS (3 × 5 min). In order to prevent non-specific binding, slices were pre-incubated with blocking buffer (1% bovine serum albumin (BSA) and 0.3% Triton X-100 in PBS) for 2 h at room temperature. Slices were incubated with Iba1 antibody (WAKO, 019-19741; 1:1000), mouse Ab-T1 (0.5 μg/ml), or anti CD68 (Santa Cruz Biotechnology, sc-20060 AF488; 1:200) overnight at 4 °C. Slides were rinsed with 0.3% Triton X-100 in PBS (3 × 5 min) followed by incubation with Cy5 goat anti-Rabbit IgG (Abcam, ab6564; 1:200) or Alexa Fluor 488 anti-Mouse IgG (Abcam, ab150113; 1:200) in blocking buffer for additional 2 h at room temperature. Slides were washed 3 times in 0.3% Triton X-100/PBS and a drop of mounting buffer (DAPI Fluoromount-G. Cat#: 0100-20, SouthernBiotech) was added before visualized on a confocal laser scanning microscope (TCS SP8, Leica).

4-well glass slides were pre-coated with Coll-I and HBEC cells seeded and incubated as described. Cells were fixed, blocked, and stained with FITC-conjugated anti-integrin or mouse IgG1 isotype control antibody. MMP-1 was stained with a rabbit anti-human MMP-1 primary antibody (Millipore) and Cy5-goat anti-rabbit IgG (Abcam) as secondary antibody. Staining with secondary antibody alone was used as control. For F-actin staining, cells were permeabilized with 0.5% saponin (v/v) and stained with phalloidin conjugated with Alexa Fluor 594 (Thermo Fisher Scientific). DAPI was used as nuclear counterstain. Slides were scanned using a 63× oil immersion objective and to avoid bleed-through effects, each dye was scanned independently in a Leica TCS SP5 confocal microscope equipped with 405 nm diode laser, 488 nm argon laser, 543 and 633 nm HeNe lasers, and using the Leica Application Suite 2.6.2 software (Milton Keynes, UK). Images were edited using ImageJ software v1.46r (NIH, MD, USA).