Clone mib 1

Ki-67 scores were retrospectively obtained from pathology reports at initial diagnosis on the primary tumor prior to any treatment. IHC staining on formalin-fixed paraffin-embedded tissue sections was performed. Mind Bomb 1 mouse monoclonal antibody (manufactured by Dako) was used to detect Ki-67. The staining protocol included de-paraffinization (30 minutes at 72°C) and rehydration with antigen retrieval performed at 100°C for 20 minutes with Tris-EDTA buffer, pH 6.0. Endogenous peroxidase was blocked with 3% peroxide for 5 minutes. Primary anti-Ki-67 antibody (Dako, clone MIB-1) was applied at 1:100 dilution for 15 minutes. Post primary antibody detection was carried out using a commercial polymer system (Bond Polymer Refine Detection, Leica), and stain development was achieved by incubation with diaminobenzidine (DAB) and DAB enhancer (Leica). The robust quality control and quality improvement program of the IHC lab at MDA was fully applied to the anti-Ki-67 IHC assay. A positive control was added to every IHC run (reference tonsil tissue, batch control) and was reviewed by a member of the IHC medical directorship team. Records of batch control results were documented daily in internal laboratory records. Stains were evaluated by designated breast pathologists, who visually estimated the percentage of positively staining invasive carcinoma cells.

All tissue samples were histologically classified according to the WHO classification of tumors of the central nervous system.^{[19 (link)]} Additional immunohistochemistry (IHC) study was performed with antibodies against proliferation-associated antigen Ki-67 (clone: MIB-1, primary antibody dilution: 1:200; Dako) following standard protocols. IHC results of the Ki-67 index were quantified using semiquantitative staining scores according to cell and vascular density.^{[20 (link),21 (link)]}

Haematoxylin and eosin staining were performed as previously described (Maharani et al., 2018 (link)). Immunohistochemistry analysis was performed as previously described (Gulay et al., 2021 (link)) with slight modifications, as described below. Heat‐induced antigen retrieval was performed twice in Tris‐EDTA buffer (pH 9.0) in a microwave oven for 5 min. Tissues were stained with mouse anti‐Ki‐67 monoclonal antibody (Clone MIB‐1, DAKO) and mouse anti‐5mc monoclonal antibody (33D3, Abcam) overnight at 4°C. To measure the Ki‐67 labelling index, more than 1000 cells were counted in randomly selected high‐power fields, and the number of Ki‐67‐positive nuclei was recorded in each field. The labelling index was defined as the ratio of Ki‐67‐labelled cells to the total number of cells counted and was expressed as a percentage. 5mc values were calculated from OD mean value from >1000 cells in each patient analysed by QuPath ver. 0.2.1 (Bankhead et al., 2017 (link)).

After tumor resection, histopathologic and immunohistochemical analysis was performed. In this analysis, Ki67 LI was measured. Tissues were obtained from formalin-fixed (4%), paraffin-embedded blocks cut into 3-5 µm sections. All sections were mounted on poly-L-lysine coated slides and left in a 45 °C incubator overnight before staining. The slides were deparaffinized in xylene and rehydrated with a series of decreasing ethanol concentrations and finally distilled water. Antigen retrieval was performed immersing the slides in a thermostat bath with preheated 10 mmol/L citrate buffer (pH 6.0) for 40 minutes at 97 °C, and after that cooled at room temperature for 20 minutes. Then the slides were treated with 3.0% H₂O₂ in distilled water for 10 minutes, to block endogenous peroxidase activity. After blocking nonspecific antigen with normal rabbit serum for 10 minutes, the slides were incubated for 30 minutes at room temperature with anti-human Ki-67 antibody (1:200; Clone MIB-1, Code M7240; Dako, Glostrup, Denmark). Negative controls were obtained by omitting the primary antibody. Only definite nuclear staining was regarded as positive. Cases were scored by the percentage of tumor cells that stained, determined in more visual fields with the most intense staining (hot spot).