Chemmate envision kit

Immunohistochemical analysis was performed as previously described [41 (link),44 (link)]. Briefly, paraffin-embedded mouse liver tissues were sectioned, and the sections were placed on microscope slides. The deparaffinized sections were boiled in buffered sodium citrate solution (0.01 mol/L, pH 6.0) for 10 min and subjected to immunohistochemical staining with anti-Ki67 antibody (ab16667, 1:200; Abcam) and anti-GFP antibody (ab6673, 1:400; Abcam) followed by horseradish peroxidase-linked secondary antibody for 30 min. The tissues were stained with 3,3′-diaminobenzidine using the ImmPACT DAB (Vector Laboratories, Burlingame, CA, USA) or the ChemMate EnVision Kit (Agilent Technologies, Inc., Santa Clara, CA, USA). Slides were immersed in hematoxylin for counterstaining and then observed under a Nikon Biophot microscope (Japan) and photographed using a digital camera (Nikon Digital Sight DS-Ri1, Tokyo, Japan).

Immunohistochemistry was conducted as previously described [30 (link)]. Briefly, paraffin-embedded xenograft tumor tissues were sectioned and placed on glass microscope slides. After the sections were deparaffinized, they were boiled in buffered sodium citrate solution (0.01 mol/L, pH 6.0) for 10 min and subjected to immunohistochemical staining with anti-Ki-67 antibody (ab15580, 1:1000; Abcam, Cambridge, UK), followed by horseradish peroxidase-conjugated secondary antibody for rabbit IgG for 30 min. Then, the tissues were stained with 3,3′-diaminobenzidine using the ChemMate EnVision Kit (Agilent Technologies, Inc., Santa Clara, CA, USA). The slides were briefly immersed in hematoxylin for counterstaining and then observed under a Nikon Biophot microscope (Nikon, Tokyo, Japan) and photographed using a digital camera (Nikon Digital Sight DS-Ri1, Nikon). The photographs were taken under 400× magnification (Supplementary Figure S3A). Ki-67-positive cells were counted from five randomly selected fields (Supplementary Figure S3B).

Immunohistochemical staining was performed on representative tissue sections cut from formalin-fixed, paraffin-embedded tissues at 3-μm thickness as our previous study [24 (link)] with a few modifications. Slides were deparaffinized with xylene, rehydrated with ethanol, heated by microwave for retrieval of antigen epitopes in a 10 mM citrate buffer (pH 6) for 7 min. Endogenous peroxidase was quenched by 3% H₂O₂. Slides were washed with Tris buffered saline for 15 min and then incubated with a primary monoclonal antibody against EMP2 (1:20; HPA014711, Sigma-Aldrich), CREB1 (1:40, sc-186, Santa Cruz) and pCREB1(S133) (1:50, sc-7978, Santa Cruz), for 1 h, followed by antibody detection using a ChemMate EnVision™ kit (K5001; DAKO, Glostrup). Two pathologists (CF Li and HY Huang) blinded to clinicopathological information and patient outcomes, independently interpreted the immunostainings. The immunointensity was scored based on the extent of moderately to strongly-stained tumor cells exhibiting combined membranous and cytosolic (EMP2) or nuclear [CREB and pCREB(S133)] staining, and labeled as 0+, < 5%; 1+, ≥ 5%, but < 25%; 2+, ≥ 25%, but < 50%; 3+, ≥ 50%, but < 75%; and 4+, ≥ 75%, respectively. A specimen showing EMP2 staining less than 1+ was regarded as loss of EMP2 expression. For CREB1 and pCREB1(S133), 4+ staining were regarded as high expression.

Normal and breast tumor formalin-fixed (10%) and paraffin-embedded samples were selected for tissue microarray construction. Briefly, tissue cylinders with 1.4 mm were punched from tumor areas of each donor tissue block and placed in a recipient paraffin block (tissue microarray), using a manual Tissue Puncher/Arrayer (Beecher Instruments, Sun Prairie, WI, USA). Endogenous peroxidase on de-paraffinized sections was blocked with EnVision TM Flex Peroxidase-Blocking reagent (Dako, Glostrup, Denmark). Antigen retrieval was performed by microwaving in citrate buffer (pH = 6.0) for 20 minutes. Slides were incubated with protein block serum-free (Dako) for 30 minutes and then with anti-Arl13b antibody (Sigma-Aldrich) in EnVisionTM Flex Antibody Diluent (Dako) for 60 minutes at room temperature. The slides were washed and incubated with EnVision/HRP rabbit (ChemMate Envision Kit, Dako) and developed with 3, 3´diaminobenzidine-hydrochloride (DAB, Dako) and counterstained with Harris hematoxylin. Slides were mounted with Entellan and analyzed by light microscopy.

The Envision system (ChemMate Envision Kit; Dako, Glostrup, Denmark) was used as described by the manufacturer to stain paraffin-embedded sections for IHC analyses. The primary antibody was a rabbit polyclonal anti-NPTX2 antibody (Sigma-Aldrich, St. Louis, MA, USA; PRS4573) diluted 1:1000 with antibody diluents (Dako, Glostrup Denmark). Dewaxed and dehydrated sections in Dako Real Target Retrieval Solution, pH 9 (Dako, Glostrup, Denmark) were heated using a 2100 retriever (Aptum Biologics, Ltd., Southampton, UK) for antigen activation. Staining scores were calculated as the sum of percentage and intensity scores, with scores > 4 indicating NPTX2 overexpression (Table A3).
IHC scores were independently evaluated by two researchers (KM and KKo).

Oral tissues were fixed by neutral-buffered formalin and embedded in paraffin to assess SMAD2 phosphorylation. After preparation of thin sections, slides were deparaffinised with xylene, followed by rehydration through graded alcohol to water. Antigen retrieval was performed in target retrieval solution (pH 9.0) at 120 °C for 15–20 min (S2367;  Dako). Then, endogenous peroxidase was quenched with 3% (v/v) H₂O₂ for 5 min. After blocking with 1% bovine serum albumin (BSA) at room temperature for 2 h, the sections were incubated with a primary antibody diluted with 1% BSA in phosphate-buffered saline (PBS). Slides were incubated overnight at 4 °C with primary monoclonal antibodies. Primary antibody was as follows: anti-phospho-SMAD2 (1:100, 3108; Cell signalling Technology). Slides were then incubated with horse radish peroxidase using a ChemMate EnVision kit (Dako) for 2 h and washed twice with PBS. Immunoreactivity was visualised with 0.6 nm 3,3′-diaminobenzidine tetrahydrochloride (Dojindo) and counterstained with haematoxylin. Images were acquired with a BX53 microscope and DP72 microscope digital camera (Olympus) and analysed using Olympus cellSens software. ImageJ software was used to quantify phosphorylated-SMAD2 (p-SMAD2) protein levels.