Later, they were washed in phosphate buffer saline (PBS) and finally incubated with PBS buffer containing DCFH-DA (Sigma-Aldrich, Taufkirchen, Germany) for 30 min.
Virus-positive cells were detected using a mouse monoclonal antibody that recognises the NP protein of influenza A viruses (9G8 Abcam), and a goat anti-mouse IgG-alkaline phosphatase conjugate (Sigma), both in buffered saline containing 0.1% v/v Tween, and finally incubated with an alkaline phosphatase substrate (NBT/BCIP in TMN buffer; Sigma).
After lysis, the inclusion bodies were washed thoroughly with buffer containing 1% Triton X-100, followed by 1% sodium deoxycholate wash and finally incubated overnight in unfolding buffer containing 6M guanidinium hydrochloride (GdmHCl).
Glutaraldehyde (GTA, 2.5%) in phosphate buffer pH 6.0 was subsequently coupled to the support for 1 h and finally incubated with peroxidase for 24 h at 4°C.
Cells were washed and fixed with 3.7% paraformaldehyde in PBS, permeabilized with PBS 0.2% BSA, 0.05% Saponine buffer for 30 min at 4°C, incubated with the 6E9 mAb for 30 min at 4°C, and finally incubated with Texas Red anti-mouse IgG secondary antibody (Vector Laboratories, Burlingame, CA).
Extraction buffer was added to each sample, which again was frozen in liquid nitrogen, thawed at 42 °C, disrupted and finally incubated at 42 °C for 90 min, followed by subsequent RNA isolation.
Sections were serially incubated with goat anti-choline acetyltransferase (ChAT) (Millipore; 1 2000 in TNB blocjing buffer) at 4°C overnight, incubated with HRP-conjugated donkey anti-goat IgG (Abcam, 1 2000) for another 1 h at room temperature and finally incubated with tetramethylrhodamine plus amplification tyramide reagent (1 250 in amplification solution) for 20 min at room temperature.
After three successive washings with buffer, the wells were finally incubated for 1 hour with diluted horseradish peroxidase-conjugated antibody (dilution 1 1,000 in washing buffer) before the addition of 100 μl of developing solution (3,3',5,5'-tetramethylbenzidine 3,3',5,5'-tetramethylbenzidine1% dimethylsubstratee).
After three washings in LS-buffer the membranes were finally incubated with a horseradish peroxidase-conjugated goat anti-rabbit antibody (1∶50000).
Membranes were finally incubated with Detection Buffer (0.1 M Tris-Cl, 0.1 M NaCl, pH9.5) and CSPD (Roche) for 5 min and exposed to X-ray films for 4 hours or overnight.
