Envision detection system k5007

Immunohistochemical staining was performed using the two-step EnVision method (Dako, Glostrup, Denmark). Paraffin-embedded tissues were cut into 5-μm serial sections, transferred onto adhesive slides, and dried at 65°C for 2 hours. The sections were deparaffinized with xylene and rehydrated through graded alcohols. Endogenous peroxidase activity was blocked with 0.3% hydrogen peroxide solution for 30 minutes at room temperature and antigen retrieval was performed at 100°C for 30 minutes in citrate buffer (10 mmol/L; pH 6.0). After washing three times with PBS at 5 minutes each time, the sections were incubated with 10% normal goat serum to block nonspecific binding. Then, the sections were incubated with rabbit anti-human B7-H4 monoclonal antibody (1:400; clone number EP1165; Abcam, Cambridge, MA, USA) at 4°C overnight, followed by immunodetection using the Dako EnVision detection system (K5007). The slides were counterstained with Mayer's hematoxylin, dehydrated in graded alcohol, and mounted with a neutral resin. The negative control was performed by replacing the primary antibody with PBS. Human tonsil tissue was used as the positive control.

Specimens were fixed in 10% neutral buffered formalin solution, embedded in paraffin, serially sectioned at 4 μm, and then stained with CDC6 monoclonal antibody (ab109315) or antibodies such as anti-CD20, CD79a, CD3, CD5, CD10, BCL-6, MUM1, BCL-2, Ki-67, CyclinD1 by the EnVision Detection System K5007 (Dako, Denmark) according to the manufacturer’s instructions. Phosphate buffered saline (PBS) was used as a negative control. All specimens were reviewed and confirmed independently by two pathologists. Breast cancer tissue was used as CDC6 positive control. To quantity CDC6 expression, five high-power fields (x200) were randomly selected from each section, and 500 cells were counted and scored according to the immunoreactive score method. The presence of brownish-yellow granules in the nucleus or cytoplasm was recorded as CDC6 positive. Based on the percentage of positive cells, 0 point was assigned for < 25%, 1 point for 25-50%, 2 points for 50-75%, and 3 points for > 75%. The staining intensity was also categorized, with 1 point was assigned for no color, 2 points for light yellow, 3 points for brownish-yellow, and 4 points for dark brown. Both scores were multiplied to calculate a final score from 0 to 12, with < 6 being negative expression and ≥ 6 being positive expression.

Paraffin sections of 4 micrometers were dried overnight at 37 °C and then for one hour at 60 °C before deparaffinization and rehydration. Heat induced epitope retrieval (HIER) was performed in target retrieval solution (TRS) pH6 for 20 min at 97 °C and then cooled to 65 °C, conducted by PT Link (DAKO Denmark A/S, Produktionsvej 42 DK-2600 Glostrup, Denmark). DAKO Autostainer Plus was used for immunodetection. After incubation of primary antibodies, sections were treated for 10 min in Dako REAL Peroxidase Blocking Solution (S2023) to prevent endogenous peroxidase activity. Secondary antibodies were incubated for 30 min (HRP Rabbit/Mouse EnVision—Polymer, Dako REAL Envision Detection System K 5007). DAB+ Chromogen (Dako REAL Envision Detection System) was utilized for 10 min before conducting hematoxylin counterstain. Lastly, sections were dehydrated and coverslips applied. Positive controls of duodenum, stomach, pancreas and pituitary gland were treated in the same manner as tumor samples. The primary antibodies were omitted for negative controls. Details on antibodies are listed in Table 1. TMA cores with <50% remaining tissue were excluded.