K005 ap red detection system

Immunohistochemistry was performed on formalin-fixed and paraffin-embedded tissue via the avidin-biotin-complex-method using the K005 AP/RED Detection System (Dako, Glostrup, Denmark). The following antibodies were used: monoclonal antibodies against brachyury (H-210, Santa Cruz, Dallas, USA, 1:100), epithelial membrane antigen (EMA; E29, Dako, 1:500), vimentin (VIM3B4, Dako, 1:300), cytokeratin (AE1 + AE3, Dako, 1:100), Ki-67 (MIB-1, Dako, 1:200), p53 (DO-7, Dako, 1:500) and the polyclonal antibodies against S100-protein (Dako, 1:1000) and HOXA10 (OriGene Technologies, Rockville, USA, 1:100). Appropriate positive and negative controls were included.
The ratio of positive chordoma cells was characterized as follows: “no immunoreactivity detected” (−), “immunoreactivity in up to 30% (+), “immunoreactivity in more than 30% and up to 70%” (++) and “immunoreactivity in more than 70%” (+++) of the total number of chordoma cells in the section^{53 (link)}.

Immunostaining of formalin-fixed and paraffin-embedded cell blocks and tissue sections (4 μm) were carried out by the avidin-biotin-complex method applying the K005 AP/RED Detection System (Dako, Glostrup, Denmark). The antibodies used are given in Additional file 1 (Additional Table 1). The ratio of positive cells was annotated as follows: “no immunoreactivity detected” (−), “immunoreactivity ≤30%” (+), “immunoreactivity >30% and <70%” (++), “immunoreactivity ≥70%” (+++).

Immunohistochemical staining was performed through the use of the avidin–biotin complex (ABC) method, applying the K005 AP/RED detection system (Dako, Glostrup, Denmark) on 4 µm-thick sections of formalin-fixed and paraffin-embedded cell as well as tissue blocks.
The primary antibodies used were rabbit monoclonal anti-PTEN (138G6, Cell Signaling, Danvers, MA, USA, 1:100) and p16INK4a (1D7D2, Invitrogen, Carlsbad, CA, USA, 1:400). Immunostainings were evaluated by an experienced pathologist who was blinded to the clinicopathological parameters and clinical outcomes of the patients.