K8020

Formalin-fixed and paraffin-embedded
(FFPE) tissues were cut into 3–4 μm sections and put
on FLEX IHC microscope slides (K8020, DAKO). Slides were heated at
60 °C for 60 min and deparaffinized in xylene (2 × 10 min).
Rehydration was performed in decreasing concentrations of ethanol
(100% ethanol: 1 × 5 min, 95% ethanol: 1 × 5 min) followed
by rinsing in distilled water. The immunohistochemical (IHC) staining
for KI67 was performed using an Autostainer Plus (DAKO) instrument.
Antigen retrieval was performed on a PT-LINK (Agilent) instrument
using the EnVision FLEX target retrieval solution (pH 9, dilution:
1:10) at 98 °C for 20 min. Slides were stained by incubating
the primary antibody (Ki67:clone MIB-1, M7240, Agilent Technologies)
at the following dilution: 1:200 (temperature: RT, time: 30 min).
The antibody–antigen complex was visualized using the EnVision
FLEX DAB detection kit (K801021-2, Agilent Technologies) and counterstained
with Mayer’s hematoxylin (S3309, Agilent Technologies). Stained
slides were dehydrated in increasing concentrations of ethanol (95%
ethanol: 1 × 3 min, 100% ethanol: 1 × 3 min), followed by
xylene (2 × 5 min). Cover glasses were mounted using a Coverslipper
DAKO (Agilent Technologies), and slides were left to dry prior to
staining evaluation.

From 171 tumor specimens, 3-μm thick FFPE sections were freshly cut, mounted on microscopic slides ([K8020] Dako, Glostrup, Denmark) and air dried at 65 °C for 30 minutes. BRAF IHC analysis was done on a Dako Autostainer Link 48 system. In brief, antigen retrieval was performed with EDTA (pH 9) using the PT link (Dako) for 10 minutes at 97 °C, subsequently followed by 5 minutes blocking with EnVision FLEX Peroxidase-Blocking Reagent, 20-minutes primary antibody incubation with “Mouse Anti-Human BRAFV600E Monoclonal Antibody VE1 (E19292—Spring Bioscience [1:50 dilution]), 15 minutes incubation with EnVision FLEX + Mouse (Linker) and 20 minutes incubation with Envision FLEX HRP-labeled polymer. Visualization was performed using chromogen substrate, either DAB for 10 minutes or AEC (for heavily pigmented tumors) for 20 minutes and tissues were counterstained with hematoxylin. A mutant control (BRAF c.1799 T > A [p. V600E]) as determined by pyrosequencing and a wild-type control (BRAF wild type as determined by pyrosequencing) were included in each staining procedure.