Anti ki67 antibody

Immunohistochemistry analysis of tumour samples was performed using the following primary antibodies: mouse monoclonal anti-α-SMA antibody (A5247, 1:1000, Sigma-Aldrich), anti-Ki-67 antibody (M7240, 1:200, Dako, Agilent Technologies, CA, USA) and phospho-STAT3 rabbit antibody (#9145, 1:200, Cell Signaling Technology). The amount of α-SMA- and Ki-67-positive cells were measured using the ImageJ 1.51 image analysis software (National Institute of Health, MD, USA). The number of phospho-STAT3-positive cells was counted under the microscope.

After 24 h of acoustic levitation, the spheroids were collected and immediately fixed overnight in paraformaldehyde 4% (Alfa Aesar, Kandel, Germany) diluted in PBS (Eurobio Scientific, Les Ulis, France). They were then embedded in a fibrin gel to facilitate manipulation for preparing samples. The fibrin gel containing the spheroids was included in paraffin and $5\mathrm{\mu }\mathrm{m}$ thick sections were cut. The slices were stained with H&E for general structure. Ki67 immunohistochemistry was performed with anti-ki67 antibody (Agilent-Dako, Santa Clara, United States) 3-step immunoperoxidase technique.

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)].

Paraffin-embedded 4% PAF-fixed rat colons were sectioned at 5 μm. The sections were stained with Hematoxylin Phloxine Saffron and an average of 12 crypt depths was measured per animal. For immunohistochemistry, the sections were immuno-labeled with mouse monoclonal anti-GLP-1 antibody ([8G9] Abcam) and rat monoclonal anti-Ki67 antibody (DAKO, Les Ulis, France) using a detection kit (Bond Polymer Refine detection; DS9800; Leica Microsystems). The primary antibody was substituted by PBS as a negative control. The number of GLP-1-positive cells was determined by counting positive cells per μm² (one labeled slide per animal) of mucosa and the number of Ki67-positive cells was determined by counting the positive cells per crypt (10 per animal) using Calopix image analysis software (TRIBVN, Chatillon, France). The results are expressed as the number of GLP-1-positive epithelial cells per μm² and as the percentage of Ki67-positive epithelial cells per crypt.

Immunohistochemical staining was performed as described previously^{18 (link)}. Anti-HER2 antibody (OP15) was from EMD Millipore, anti-ER antibody (RM9101) was from ThermoFisher, and anti-Ki67 antibody was from DAKO (Carpinteria, CA).