For this reason it would be useful to obtain an automatic segmentation and enrichment of lines in order to apply to each section the best algorithm and the appropriate parameter.
Treatments (distilled water, isopropyl alcohol, 3% hydrogen peroxide, 2% l-lactic acid, 10% povidone-iodine, and 1% cetylpyridinium chloride (CPC)) were then applied to each section and the sections were sampled for enumeration of aerobic plate counts (APCs), coliforms, and Escherichia coli.
Alexa Fluor secondary antibodies (Molecular Probes) were used at a 1/200 dilution in 10% ADB and applied to each section.
Tissue sections were then washed in washing buffer and DAB-chromogen/substrate mixture (DAKO, Hamburg, Germany) was applied to each section.
At this point, the anti-NVP antiserum was diluted to 1 100 in 10% goat serum and applied to each section overnight.
After rinsing, 100 μl of HRP Polymer Conjugate was applied to each section, and incubated for 10 min, followed by a rinse with PBS.
Following incubation with the indicated primary antibodies, Alexa Fluor 555-conjugatedonkeyey anti-rabbit (Molecular Probes, cat A-31572) secat A-31572ibodiesecondaryplied to eantibodiesn.
Finally, 100 μl of DAB chromogen was applied to each section and incubated for 3 10 min. Samples were then rinsed well with distilled water.
Sections were then washed twice with PBS and either the primary antibody or the normal goat or rabbit IgGs (negative control) was applied to each section and left at 4 °C overnight.
Sections were then washed twice with phosphate buffered saline (PBS) and 1 ml of either the primary antibody or the normal goat or rabbit IgGs (negative control) was applied to each section, and left at 4°C overnight.
Each sample was washed twice in 1 × PBS in NEBuffer 3, and 50 μL of the same buffer containing 100 units/mL of CIAP was applied to each section before incubating for ≥ 2 h.
