Envision flex rabbit

For immunohistochemical analyses specimens were placed in ice-cold isopentane, stored at -80°C and followed by standardized procedures to produce paraffin blocks and paraffin sections of 2 μm thickness. Immunohistochemistry was performed according to standardized methods in a fully automated manner (Dako Autostainer plus, Dako, Germany) by use of a 1:400 dilution for the first rabbit anti-ChAT antibody (Abcam ab68779, UK; second ab: Envision Flex+rabbit, Dako K8019, Germany). Histology was evaluated and documented using a Keyence BZ-9000E Hs all-in-one microscope (Keyence, Germany). For immunogold transmission electron microscopy specimens were fixed in buffered formaldehyde (3.7%) for 2 h at room temperature and stored overnight in phosphate-buffered saline at 4°C. Fixed specimens were embedded in epoxide, followed by the use of a standard protocol with application of the first antibody (ab68779) in a dilution of 1:100 and the second gold-labeled anti-rabbit antibody (Sigma Aldrich, G7402) in a dilution of 1:20. For scanning electron microscopy specimens were fixed in 2.5vol% glutaraldehyde (6 hours at room temperature). The analyses and photo-documentation were performed by using a transmission electron microscope (JEM-1400, Jeol, Tokio, Japan). Negative control experiments without the primary antibody were always carried out in parallel.

From the TMAs 2 µm-thick sections were cut and mounted onto glass slides. All glass slides were stored for 2 days at 58°C at the drying chamber, deparaffinized using xylene and dehydrated with ethanol. Subsequently all TMAs were stained using ready to use antibodies for vimentin (monoclonal mouse anti-Vimentin, DAKO, clone V9, code IR630), cytokeratin 7 (monoclonal mouse anti-human Cytokeratin 7, DAKO, clone OV-TL, code IR61961) and histone H2A (monoclonal rabbit anti-human H2A.X(D17A3)XP, CellSignaling, code 7631). Host dependent Streptavidin–biotin based peroxidase detection was performed using the EnVision^® Flex Peroxidase-Blocking Reagent (DAKO, SM801), EnVision^® Flex + Rabbit (LINKER) (DAKO, SM804) or EnVision^® Flex + Mouse (LINKER) (DAKO, SM805) and EnVision^® Flex/HRP (DAKO, SM802). Counterstaining was performed with hematoxylin before adding a cover slip. For external positive controls, tissue specimens derived from the colon, placenta and kidney were added onto the TMA.