Anti mouse igg fitc

We collected 100 μL culture medium (= total 1.2 × 10⁵ Sf9 cells) from a total of 1.2 × 10⁷ infected Sf9 cells in a 75 cm² flask (10 mL culture medium/flask). Additionally, we collected 100 μL culture medium (= total 3.7 × 10⁵ 293F cells) from ~1.1 × 10⁸ transfected 293F cells in a 125 mL Erlenmeyer flask (30 mL culture medium/flask). The cells were permeabilized on the glass slide with 100% cold acetone. Subsequently, the fixed cells were probed with anti-HPV16 L1 antibody CAMVIR-1 (Abcam, Cambridge, UK) and captured with anti-mouse IgG-FITC (Sigma, St. Louis, MO, USA). Immune-stained cell monolayers were thoroughly washed with PBS and covered with mounting medium with DAPI (Abcam). The immunofluorescence images were inspected under an inverted microscope at 40× magnification. Transfection efficiency was determined by the ratio of FITC (green)-positive cells to DAPI (blue)-stained cells.

We determined the location of nucleoli by immunostaining interphase nuclei with a fluorescently labeled antibody against fibrillarin, the basic component of the nucleolus fibrillar domain [20 (link)]. The material was extracted in the EBR solution (0.13M NaCl, 0.04M KCl, 0.018M CaCl₂, 9mM HEPES) at +4°C and fixed in 4% of paraformaldehyde for 20 min at room temperature. Fixative solution was washed away by 1×PBS at room temperature three times for 5 min, and tissues were treated with the PBSTr solution (0.3% Triton-X100 in 1×PBS) for 30 min at room temperature. Then the material was incubated in block buffer (BB) (4% powdered milk, 10% FBS) for 30 min and then shaken in 1% solution of primary antibodies (Anti-fibrillarin [38F3], Abcam, Cambridge, UK) in BB at +4°C during the night. After that, tissues were washed in PBSTr three times for 15 min at room temperature. Staining was conducted in 0.25% solution of secondary antibodies Anti-Mouse IgG—FITC (Sigma-Aldrich, Dia-m, Novosibirsk, Russia) in BB at +4°C overnight. The washing step was performed in the same manner as above. Finally, the tissue was stained by DAPI (Prolong Gold Antifade, ThermoFisher Scientific, Dia-m Company, Novosibirsk, Russia) at +4°C for 8 hours.

WEB2086 was purchased from Tocris Bioscience (UK). Human TNFα ELISA kit was purchased from R and D systems Europe. PAF ELISA kit was purchased from Blue Gene Biotech, China. LPS, antimonoclonal anti-human CD62, anti-mouse IgG FITC, and mouse IgG1 were purchased from Sigma-Aldrich (Poole, UK). MSU crystals were purchased from Enzo Life Sciences, Germany. HUVEC cells were purchased from ATCC cell lines, UK. Recombinant human PAF-acetylhydrolase was purchased from Sigma-Aldrich, USA.

Reagents were as follows: BK, 40 kDa FITC-Dextran, hyaluronidase, arachidonic acid (AA) anti-Mouse IgG FITC, anti-Rabbit IgG TRICT, anti-B2 receptor, and anti-β-actin (Sigma Chemical); anti-COX-2 and anti-mPGES-1 (Cayman Chemicals); IKK inhibitor VII, anti-p65 and anti CD31 (Millipore); anti-ZO-1 (BD Transduction); anti-p-β catenin (Ser33, 37/Thr41) and anti-β catenin (Cell Signaling); anti VE-Cadherin (e-Bioscience); anti-H2A (Santa Cruz), anti-phospho-p65 (Bioss); anti-CD133 (Boster Immunoleader). Fasitibant ((4S)-4-amino-5-{4-[4-(2,4-dichloro-3-{[(2,4-dimethylquinolin-8-yl) oxy]methyl}benzenesulfonamido)oxane-4-carbonyl]piperazin-1-yl}-N,N,N-trimethyl-5-oxopentan-1-aminium chloride dihydrochloride or MEN16132) (batch number 2010/02), was synthesized in Menarini Ricerche, (Chemistry Development Department, Pisa, Italy).

For immunofluorescence MitoTracker-stained cells were fixated, residual PFA was quenched with NH₄Cl (50 mM) in PBS for 30 min, cells were permeabilized with Triton X-100 (0.5% v/v) and, after washing, blocked according to the manufacturer’s instructions. The cells were covered with the antibody (Drp1: Cell Signalling, #8570, 1:50 dilution, 1 h at RT; α-Tubulin, Sigma Aldrich, St. Louis, Missouri, USA, T5168, 1:500, 1 h at RT), washed and incubated with the secondary antibody (Anti-Rabbit IgG-FITC, Sigma-Aldrich, #F9887, 1:100, 45 min at RT; anti-Mouse IgG-FITC, Sigma-Aldrich, F9137, 1:100, 1 h at RT), then mounted and analysed by fluorescence microscopy (λ_exc. = 470 ± 20 nm, λ_em. = 525 ± 25 nm).

The eWAT (epididymal WAT) was fixed and washed with phosphate-buffered saline (PBS). The sections were exposed to a primary antibody against UCP-1 (sc-518024, Santa Cruz Biotechnologies Inc., CA, USA) for 2 h at room temperature. Following incubation with a primary antibody, the sections were washed thrice for 5 min in PBS. Later, the sections were labeled using anti-mouse IgG- FITC (Sigma, St Louis, MO, USA) for 1 h at room temperature. The sections were washed thrice for 5 min in PBS, and glass coverslips were mounted with 20 μL aqueous-mount solution (Scytek laboratories, Logan, UT, USA). Images were captured using an inverted confocal microscope (Leica DMIRE2; Leica Microsystems, Wetzlar, Germany) with a 63× oil immersion objective lens. All the images were captured with the same laser intensities.

The cells were permeabilized on the glass slide with 100% cold acetone. Subsequently, the fixed cells were probed with anti-HPV16 L1 antibody CAMVIR-1 (Abcam, Cambridge, UK) and captured with anti-mouse IgG-FITC (Sigma). Immune-stained cell monolayers were thoroughly washed with PBS and covered with mounting medium with DAPI (Abcam). The immunofluorescence images were inspected under an inverted microscope at 40× magnification. Transfection efficiency was determined by the ratio of FITC (green)-positive cells to DAPI (blue)-stained cells.