Envision dual link system

Consecutive 3-μm sections were cut from each block and subjected to immunohistochemical staining with the EnVision+DualLink system (Dako, Carpinteria, CA, USA). An immunoperoxidase technique was applied following antigen retrieval with microwave treatment (95 °C) in citrate buffer (pH 6.0) for 45 min. Anti-PKM1 (Abgent, San Diego, CA, USA), anti-PKM2 (Cell Signaling Technology, Danvers, MA, USA), HIF1α (Thermo Fischer Scientific, Waltham, MA, USA), and anti-Ki-67 antibody (DAKO) diluted at 0.5 μg/mL were used as primary antibodies. After 2 h of incubation at room temperature, the sections were incubated with a secondary antibody for 30 min. The specimens were color-developed with diaminobenzidine (DAB) solution (Dako) and counterstained with Meyer’s hematoxylin (Sakura Finetek, Tokyo, Japan). Immunostaining of all samples was performed using the same conditions of the antibody reaction and DAB exposure. Immunoreactivities of PKM1 and PKM2 were classified according to the Allred’s score (AS) [50 (link)]: grade 1 for AS = 0, 2 for AS = 2–4, and 3 for AS = 5–8. Grade 2 and 3 cases were regarded as immunopositive [5 (link)]. We also observed 20 microscopic fields per case at 200× magnification and counted the tumor cells per case; the results were expressed as the percentage of tumor cells, with positive nuclei defined by Ki-67 and HIF1α LI [10 (link)].

After deparaffinization, heat-induced epitope retrieval was performed on a PT Tissue Link system (Histolab). IHC for collagen type IV, periostin, and decorin was performed using the EnVision Dual Link System (K4065, Dako, Glostrup, Denmark) according to manufacturer’s instructions, including horse-radish peroxidase (HRP)-coupled secondary antibodies and counterstaining with Mayer’s hematoxylin to visualize nuclei. IF for collagen type VI, biglycan, versican, vimentin, and vinculin was performed by incubation with primary antibodies for 1 h and with fluorochrome-coupled secondary antibodies (Thermo Fisher Scientific, 1:200) for 45 min. Sections were mounted with ProLong Gold Antifade Mountant with DAPI (Invitrogen) to visualize nuclei. Unspecific staining of the secondary antibodies was assessed by omitting the primary antibodies (negative control). For antigen retrieval and antibody-specifications see Table 2.

Immunohistochemistry was performed as described previously [12] (link), [13] (link). Briefly, formalin-fixed paraffin sections were incubated with anti-FOXM1 antibody (NBP1-30961, 1∶40, Novus Biologicals, CO, USA) at room temperature overnight and stained using EnVision+ Dual Link System (K4061; Dako, CA, USA). Antigen retrieval was performed using EDTA buffer, pH8.0, in a pressure cooker for 30 min. All sections were assessed by two independent investigators. The immunoreactivity of FOXM1 antibody, the intensity of stained cells and their percentages were measured in terms of intensity and percentage scores respectively. The percentage score ranged from 0 to 4: 0 = <5% of positively stained cells, 1 = 5–25% of positively stained cells, 2 = 26–60% of positively stained cells, 3 = 61–85% of positively stained cells, and 4 = 86–100% of positively stained cells. Immunohistochemical (IHC) score (from 0 to 16) was calculated by multiplying the intensity score (0–4) and the percentage score (0–4), with a maximum score of 16 [12] (link), [13] (link). FOXM1 nuclear and cytoplasmic immunoreactivities were scored separately.