Binding was performed by adding 100 μL of diluted antiserum into microwells and incubating for 1 h at room temperature, followed by thorough washes.
Diluted antiserum was then applied to the sections (in a volume sufficient to cover the tissue) and the slides were processed as previously described.
The assays were performed by mixing PRV SC and HeN1 strains, respectively, with 50 μL of serially diluted antiserum and 100 TCID50 Bartha-K61 vaccine.
Appropriately diluted antiserum to 17 β-estradiol and testosterone (New England Nuclear Corp., Boston, MA) and 0.1 mL of [3H]-steroids without antiserum (to determine nonspecific binding) were included in every assay.
Absorption controls were performed by adding an excess amount of antigen (10 100 μg of synthetic peptide diluted in antiserum) before exposure.> -wrap-foot> *C-terminal flanking peptide of NPY (used for the detection of NPY-containing neurons).
Briefly, wells were coated with 100 μL of a 1 8,000 dilution of hyperimmune rabbit antiserum diluted in PBS (except for Syd3, for which a 1 3,000 dilution was used), and the plates were incubated overnight at 4°C.
The same technique was used for the detection of collagenase-induced cleavage of Col II by using diluted rabbit antiserum (1 200) raised against the C1,2C neoepitope and diluted mouse ascitic fluid (1 200) raised against the C2C neoepitope.
After this, 100 μl pooled patient antiserum (diluted 1 500 with dilution buffer from the Anti-Gliadin IgG kit, Biohit Oyi, Helsinki, Finland) was added.
Primary antibodies were applied over night at 4°C as follows: anti-CT166 antiserum, diluted 1∶500 in blocking solution; mouse anti-Rac1 (MilliporeMillipore, Billerica, USA), diluted 1∶1000 in 0.05% TBST; mouse anti-RhoA (26C4; Santa Cruz Biotechnology, Santa Cruz, CA, USA), diluted 1∶300 in 0.05% TBST; and mouse anti-beta-actin (AC-40, Sigma), diluted 1∶2000 in 0.05% TBST.
Afterwards, the nitrocellulose membranes were incubated for 2 h with the anti-His-DUF1542 anti-His-DUF1542 anti-His-DUF15423% BSantiserum
