Immunohistochemistry for Neurofilament

For cellular analysis of NF in the ischemic brain, we used postmortem brain tissue from two patients that died from a stroke. One patient was a 59-year-old man with a >7-day-old infarct in the right parietal lobe, and the other patient was a 74-year-old female with a 3–7-day-old infarct in the left temporal lobe. Parallel tissue sections have been part of previous studies on surfactant protein-D, IL-1, and TNF (20–23). Tissue blocks containing the infarcted brain tissue were embedded in paraffin, and 2-μm-thick sections were cut on a microtome. Immunohistochemical staining was performed as routinely done at the Department of Pathology, OUH, using a fully automated VENTANA BenchMark ULTRA immunostainer (Ventana Medical Systems, Tucson, AZ, USA). The immunoreactive product was visualized using the OptiView DAB detection kit (Ventana Medical Systems), and the signal was enhanced with the Ventana Amplification Kit (Ventana Medical Systems). The sections were heated to 75°C for 4 min, deparaffinized at 72°C, and demasked using a T-EG-based buffer (10 mM Tris + 0.5 mM EGTA, pH 9.0). Sections were blocked for endogen peroxidases using 1.5% H₂O₂ in Tris-buffered saline (TBS) and incubated with monoclonal mouse anti-NF (phosphorylated and non-phosphorylated NF-H chain) antibody (clone N52, 1:1,000, Sigma-Aldrich) for 1 h. Sections were counterstained with Hematoxylin II (Ventana Medical Systems) using the BenchMark Ultra immunostainer. Finally, sections were rinsed, dehydrated, and coverslipped using a Tissue-Tek Film coverslipper (Sakura, Alphen aan den Rijn, The Netherlands).