Anti ki67

Western blot reagents were obtained from GE Healthcare (Pittsburgh, PA, USA). Fluorescent probes for caspase activity, caspase inhibitors, and annexin V assay kit were from BD Biosciences (San José, CA, USA). Culture media were from Lonza (Basel, Switzerland). MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide) and p38MAPK inhibitor (SB202190) were obtained from Sigma-Aldrich (St. Louis, MO, USA). Primary monoclonal rabbit antibodies against caspase 8 (dilution, 1:1,000; #4927), cleaved-caspase 9 (dilution, 1:1,000; #7237), MMP-2 (dilution, 1:1,000; #4022), MMP-9 (dilution, 1:1,000; #3852), p-p38 (dilution, 1:1,000; #9211), p38 (dilution, 1:1,000; #9212), and TIMP-1 (dilution, 1:1,000; #8946) were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA). Caspase 3 (dilution, 1:1,000; sc-7148), Bid (dilution, 1:1,000; sc-11423), Bcl-2 (dilution, 1:1,000; sc-783), Bcl-xL (dilution, 1:1,000; sc-634), and Bax (dilution, 1:1,000; sc-526) were purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA), and β-actin (dilution, 1:5,000; #A5441) was obtained from Sigma-Aldrich (St. Louis, MO, USA).
Anti-Ki67 (dilution 1:200) and Click-iT Tunel colorimetric IHC detection kit were from Thermo Fisher (Waltham, MA USA). DAB kit was provided from Vector laboratories (Burlingame, CA, USA).

Formalin-fixed, paraffin-embedded sections were dewaxed. Heat induced epitope retrieval was performed in a water bath containing citrate buffer (Dako North America, Carpinteria, CA, USA), blocked with 1% hydrogen peroxide and then treated for 30 min with CAS-Block (Invitrogen, Carlsbad, CA, USA) before primary antibody incubation. The primary antibodies were rabbit monoclonal anti-Ki-67 (1:200 dilution; Thermo Fisher Scientific) and rabbit monoclonal anti-CD31 (1:75 dilution; Abcam). Staining signals were detected using the Starr Trek Universal horseradish peroxidase (HRP) Detection System (Biocare Medical, Pacheco, CA, USA). Immunohistochemistry slides were scanned with a 3DHITECH PANNORAMIC Midi slide scanner, and images were captured using PANNORAMIC Viewer software (3DHITECH, Budapest, Hungary).

Immunostainings for detection of Gal-3 expression were performed in MSC plated on coverslips. The cells were fixed with paraformaldehyde 4% and incubated overnight at 4°C with the primary antibody goat anti-Gal-3, diluted 1 : 400 (Santa Cruz Biotechnology, Dallas, TX, USA). On the following day, sections were incubated for 1 h with phalloidin conjugated with Alexa Fluor 488 (1 : 200; ThermoFisher Scientific) mixed with the secondary antibody anti-goat IgG Alexa Fluor 568 (1 : 1000; ThermoFisher Scientific). Proliferating cells were evaluated by KI67 staining (anti-Ki67 1 : 1000; ThermoFisher Scientific), followed by anti-rabbit IgG Alexa Fluor 568 (1 : 1000 ThermoFisher Scientific). Dead cells were stained with PI (BD Biosciences). Nuclei were stained with 4,6-diamidino-2-phenylindole (VECTASHIELD mounting medium with DAPI H-1200; Vector Laboratories, Cambridgeshire, UK). The presence of fluorescent cells was determined by observation using an A1+ confocal microscope (Nikon).

Tumors removed from the mice were fixed in 4% paraformaldehyde overnight, embedded in paraffin, sectioned at 4 μm thickness and then were stained with HE and immunohistochemistry (IHC) assay. For IHC, sections were incubated with the primary antibody at 4°C overnight and the secondary antibody at room temperature for 30 min before staining. Slides were observed using a microscope (OLYMPUS, CX41) and staining was expressed by positive area. The primary antibodies used in the assay are listed as follows: anti-Ki67 (1:100, Thermo Fisher Scientific, RM-9106-S1), anti-PTEN (1:100, #9559), anti-p-AKT (1:100, #4060), anti-p-S6K (1:100, #9206) were purchased from Cell Signaling Technology (Danvers, MA, USA).

Serial 8 µm frozen sections from LV tissue were fixed in 1% paraformaldehyde for 10 min at room temperature and post-fixed in Ethanol/Acetic acid (2:1) for 5 min at 20 °C. Sections were blocked with PBS BSA 1% for 1 h before immunolabeling with primary antibodies against: α-SMA (Abcam), and Ki67 (anti-Ki67; ThermoFischer Scientific). A fluorescent secondary antibody tagged alexa-red-555 was used against the primary antibody. Stained sections were scanned using an epifluorescence Zeiss Axiocam microscope equipped with a digital camera. The α-SMA + and Ki67 + mean fluorescence was calculated with NIH ImageJ software (NIH) in each section, comparing it with the size of the whole area.

The following antibodies were used for immunochistochemistry: anti-Foxm1 [42] (link), [45] (link), anti-SPDEF [46] (link), anti-Ki67 (Thermo Scientific) and anti-phospho-Histone H3 (pH 3) antibody (Santa Cruz).