Histofine simple stain max po system

Immunohistochemical staining was performed as described previously [16 (link)]. Briefly, specimens were fixed with 3.7% formalin and embedded in paraffin, and then sliced sequentially at a thickness of 3 μm. Samples were deparaffinized with xylene and rehydrated with graded alcohol. After rinsing with tris-buffered saline, samples were processed for antigen retrieval with EDTA buffer (pH 9.0) at 95 °C for 20 min. Samples were incubated with antibodies against Arf6 (1:100) or AMAP1 (1:200), or normal mouse IgG (1:100) at room temperature for 60 min, and the Histofine Simple Stain MAX PO system (Nichirei) was used for visualization. The coloring reaction was performed with 3,3′-diaminobenzidine (Dojin) for 5 min. Hematoxylin was used as a counterstain. A rabbit polyclonal antibody against Arf6 and mouse normal IgG was purchased from commercial sources (Arf6, Aviva; mouse normal IgG, Santa Cruz Biotechnology, Inc.).

Mouse monoclonal anti-SET7/9 antibody (clone 5F2.3, 04-0805; Merck Millipore, Darmstadt, Germany) was diluted at 1:100. Immunohistochemistry of SET7/9 protein was performed using a Histofine Simple Stain MAX PO system (Nichirei Biosciences Inc., Tokyo, Japan) and a Ventana BenchMark XT automatic immunostainer system (Ventana Medical Systems, Tucson, AZ) according to the manufacturer's protocol. We assessed the SET7/9 staining score as to both the percentage of the extent of cell staining and intensity. Briefly, immunoreactivity for SET7/9 was scored as follows; proportional score (PS): 0 (negative), 1 (1 - 5% of tumor cells), 2 (6 - 25%), 3 (26-50%), and 4 (51∼100%), and intensity score (IS): 0 (no tumor cell staining), 1 (weak), 2 (moderate), and 3 (strong). The total score (TS) was obtained by summing IS and PS. Because more than half of the tumor cells were stained in SET7/9 expression cases, TS ≥ 6 was considered to represent high expression (retained expression) of SET7/9. Thus, we made two groups, SET7/9 retained (high) and loss/weak (low).