The cells were fixed with 4% paraformaldehyde (Histolab Products

The cells were fixed with 4% paraformaldehyde (Histolab Products AB, Gothenburg, Sweden) for 10 min and washed twice with phosphate buffer saline (PBS) (Invitrogen) containing 1% BSA (PBS–BSA). The cells were permeabilised with PBS–BSA containing 0.05% saponine (PBS–BSA–Sap) for 20 min. Thereafter the cells were incubated for 1 h with a cocktail of rabbit polyclonal antibody against Toll-like

receptor, TLR4, (Santa Cruz Biotechnology, Santa Cruz, CA, USA) diluted 1:100 and a monoclonal antibody against OX42 diluted 1:100 in PBS–BSA–Sap. The cells were washed with PBS–BSA–Sap. for 3×5 min and then incubated with a mixture of FITC conjugated F(ab′)2 donkey anti-rabbit IgG and Texas Red conjugated F(ab′)2 donkey anti-mouse IgG secondary antibodies (Jackson Immuno Research, Westgrove, USA), both diluted in PBS–BSA–Sap. The cells were washed with PBS–BSA–Sap for 3×5 min and finally rinsed Selleckchem PD0325901 with PBS. Controls were treated similarly except for incubations with the primary antibodies. The cover slips were mounted on microscope slides with a fluorescent mounting medium (DAKO, Glostrup, Denmark) and viewed in a Nikon Eclipse 80i microscope. Pictures were taken with a Hamamatsu C5810 colour intensified 3CCD camera. Cells were rinsed twice in phosphate

buffered saline (PBS) and immediately lysed 20 min on ice in cold RIPA lysis buffer. The procedure was done according to the process described by Persson et al. (2005). Separate aliquots were taken for protein concentration determination. All samples were correlated for total protein contents and an equal loading Belinostat chemical structure of 20 µg total protein of each sample was applied Non-specific serine/threonine protein kinase in each lane of the gel. SDS-PAGE

were conducted using the Novex pre-cast gel system (Invitrogen) according to the manufacturer’s recommendations using 4–12% Bis-–Tris gels (Invitrogen) at 200 V for 50 min. The separated proteins were then transferred at 30 V for 60 min to a nitrocellulose membrane (Invitrogen) using NuPAGE transfer buffer (Invitrogen) supplemented with methanol and NuPage antioxidant. The membranes were rinsed twice with distilled water and the proteins were visualised with Ponceau S solution (Sigma). Proteins were blocked with 5% fat free skim milk (Semper AB, Sundbyberg, Sweden) in TBST (50 mM Tris–HCl, 150 mM NaCl and 0.05% Tween) for 60 min at room temperature. The membranes were then probed with primary antibodies overnight (+4 °C), washed 4×2 min with TBST, and subsequently probed with secondary horse-radish peroxidase (HRP) conjugated secondary antibodies for 60 min at room temperature, and finally washed several times in TBST. The primary antibody used was rabbit polyclonal TLR4 diluted 1:500. The secondary antibody used were HRP-conjugated donkey anti rabbit F(ab′)2 fragment (both from Jackson Immunoresearch) diluted 1:10000.

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