Anti ki 67 antibody clone mib 1

For immunostaining, the tumors were embedded in paraffin using standard methods. For Ki67, KRAS and PIK3CA staining, dehydrated sections were blocked with 3% BSA, and stained with anti Ki67 antibody (clone MIB-1, DakoCytomation, Glostrup, Denmark), anti KRAS antibody (clone 9.13, ThermoFisher Scientific, Schwerte, Germany), or anti PIK3CA antibody (PI3Kinase-p110α (4249, Cell Signaling Technology, Danvers, MA, USA),) processed with mouse anti rabbit-peroxidase, washed and developed according to standard methods. Counterstain was performed using hematoxylin using standard methods.
For c-MYC and KRAS immunofluorescence, dehydrated sections were boiled for 3 min in 10 mM citric acid, 0.05% Tween 20, pH 6.0, washed twice in PBS and blocked in 1.5% normal goat serum, 0.1% Tween 20 in PBS. Sections were incubated with c-MYC (clone Y69, Abcam, Cambridge, UK; 1:500) and KRAS (ab55391, Abcam, Cambridge, UK; 1:100) antibody diluted in blocking solution overnight at 4°C, washed three times in PBS/0.1% Tween 20 for 5 min, incubated with secondary antibodies (goat anti-rabbit-Cy3 and donkey anti-mouse-Alexa 488, 1:500 each) diluted in blocking solution for 1–2 h at RT, counterstained with Hoechst 33342 and mounted using Dako mounting medium.

Immunostaining was performed using 4-μm-thick FFPE sections of the same PTC cases. Anti-Ki-67 antibody (clone MIB1, Dako) was used as a primary antibody. The staining was carried out using the Dako Cytomation Autostainer Universal System (Dako) and the Envision kit (Dako) according to the manufacturer’s instruction. A single pathologist (MH) evaluated the specimens without clinical information of the patient. To obtain the Ki-67 LI, at least 500 carcinoma cells in hot areas were analyzed under × 400 magnification. Staining results were classified into three groups:<5%, 5%–10%, and >10% of positive cells.

Tissue sections were immunostained with an anti-Ki-67 antibody (clone MIB-1; 1:100; Dako, Denmark). Tissue sections were deparaffinized in xylene, rehydrated through serial dilutions of ethanol, and washed in phosphate-buffered saline (pH 7.2), and then microwaved at 500 W for 2 × 5 min in 10 mM citrate buffer (pH 6.0). After rinsing in Tris-buffered saline (pH 7.6), the sections were immersed in a solution of H₂O₂ (3%) to block endogenous peroxidase activity, followed by the addition of 5% fetal calf serum (1:20 dilution). The samples were then incubated with a Ki-67 primary antibody (12202,1:400, Cell Signaling Technology [CST]) overnight at 4°C. After a wash, the primary antibody was detected by means of an appropriate secondary antibody (Universal PV9000 Kit; Zhongshan Biotechnology, Beijing, China), incubated for 30 min at 37°C, and then visualized with diaminobenzidine (DAB). Counterstaining was carried out with hematoxylin.
Briefly, the nuclear staining intensity was assessed in 1000 cells randomly selected from 5 areas (2 distinct lesions were selected in each area) by 2 of the authors and a pathologist, under a microscope at ×400 magnification, and were graded as follows: 0, negative <10%; 1+, weakly positive 10–33%; 2+, moderately positive 34–66%; and 3+, strongly positive >66%.