Benchmark xt slide preparation system

TMA blocks consisting of a representative tumor core section 2 mm in diameter from each formalin fixed paraffin block (SuperBioChips Laboratories, Seoul, Republic of Korea) were manufactured for IHC analysis. IHC staining was performed using the BenchMark XT Slide Preparation System (Ventana) and rabbit polyclonal anti-OCT4 antibody (1:100, Abcam, Cambridge, MA, USA). Assessments of the staining were evaluated under a light microscope by two experienced pathologists who did not know the exact condition of the patient and the scores were depended on staining intensity and proportion as previously described. For each tissue core, the intensity of staining was categorized as follows: 0, negative; 1 weak; 2 moderate; and 3, strong. And based on the proportion of staining, the degree was scored on a scale of 0 (< 5%, absent), 1 (5%–25%, sporadic), 2 (25%–50%, focal) and 3 (> 50%, diffuse). The final score of each staining was obtained by multiplying the two scores. The IHC score ranged from 0 to 9, which less than 4 points was determined to negativity.

For pathological confirmation and finding various types of CTCs, immunocytochemical staining was conducted for captured cells from blood samples donated by cancer patients with RCC independently. All captured and released cells from the microfilter were gently cytospinned on slide glass and fixed with 4% paraformaldehyde in 15 minutes. After fixation step, each slide were automatically immunostained by using the BenchMark XT Slide Preparation System (Ventana Medical Systems, Inc., Tucson, AZ, USA), with antibodies to EpCAM (clone VU-1D9, 1:2,000, Calbiochem, San Diego, CA, USA), CK (clone AE1/AE3, 1:100, Dako Cytomation, Glostrup, Denmark), and CD10 (clone 56C6, 1:400, Novocastra Leica Biosystems, NewCastle, UK). Positive expression was defined as the unequivocal brownish staining in the cytoplasm and/or cell membrane. Cytologic criteria for tumor cells were as follow: large cell size (1.5 times larger than white blood cells), large nuclear size with high nuclear-cytoplasmic ratio, irregular nuclear membrane, and presence of cytoplasm^{42 (link)}. The presence of tumor cells regardless of their EpCAM, CK (AE1/AE3), or CD10 expressions was considered as CTC, and the total number of CTCs in entire slide was enumerated. All slides were examined by one pathologist (Chang, H.J.).