Rabbit anti ki67 primary antibody

Histological sections were permeabilized in 0.1% Triton v/v, 3 times for 5 min each and blocked in PBS-3% BSA w/v for 1 h and incubated with rabbit anti-Ki67 primary antibody (Abcam, Cambridge, UK), rabbit anti-α-Fetoprotein (α-FP) or rabbit α-smooth muscle actin (α-SMA) (all purchased from DAKO-Agilent, Santa Clara, CA, USA). After overnight incubation at 4 °C, sections were washed in PBS and incubated with Alexa Fluor 568 or Alexa Fluor 488 secondary antibody (ThermoFisher scientific, Waltham, MA, USA) for 1 h at room temperature and when specified stained also for TUNEL. Following extensive washes in PBS, the sections were counterstained with DAPI and mounted with PBS-50% v/v Glycerol. Images were acquired by a Nikon Eclipse Ti-S microscope equipped with a Photometric CoolSNAP EZ turbo 1394 camera.

We used Ki67 as a marker, since this is a nuclear protein known to be expressed when cells are proliferating but which remains inactive when cells enter the G₀ phase of the cell cycle (74 (link)– (link)76 (link)). The NHDF (3.5 × 10³ cells per well) were cultivated for 48 h with or without DCA in 96-well plates and were fixed using a 4% paraformaldehyde solution. Subsequently, the cells were permeabilized in 0.5% Triton X-100 in PBS and blocked with 1% bovine serum albumin (BSA) in PBS. The cells were further incubated with rabbit-anti Ki67 primary antibody (Abcam, MA, USA) and detected using Alexa Fluor 488-conjugated goat anti-rabbit antibody (Invitrogen, CA, USA). Nuclear DNA was stained with 4′,6′-diamidino-2-phenylindole (DAPI). The images were obtained using the Leica DM6000 fluorescence microscope, and 60 pictures of each cultured condition per well were taken and analyzed using the ImageJ software, which quantifies the Ki67- and DAPI-positive stained cells.

Culture media and supplements, collagenase type XI, histopaque-1077, DMSO, EDTA, IBMX, carbachol, clonidine, Accutase, Fura-2 AM, tolbutamide, LiCl, exendin-4, forskolin, agarose, bionic buffer, and BSA were obtained from Sigma-Aldrich (Dorset, UK). JTE 907 was from Tocris Bioscience (Abingdon, UK). Rabbit anti-Ki67 primary antibody was from Abcam (Cambridge, UK). cAMP HiRange and IP-one (IP₁) assays were from Cisbio (Codolet, France). HEPES, HBSS, and DAPI were from Thermo Fisher Scientific (Paisley, UK). Caspase-Glo 3/7 was from Promega (Southampton, UK). Recombinant TNFα, IFNγ, and IL-1β were from PeproTech EC (London, UK). Guinea pig anti-insulin was purchased from Dako (Cambridge, UK). Alexa Fluor 488- and Alexa Fluor 594-conjugated secondary antibodies were from Jackson ImmunoResearch Laboratories (Newmarket, UK).

IHC was performed to detect Ki-67 expression in the xenograft tumors as described previously [38 (link)]. The primary rabbit anti-Ki-67 antibody and the secondary goat anti-rabbit IgG were purchased from Abcam (Cambridge, UK). Ki-67 expression was quantified using the Image-pro-plus (IPP) software (Media Cybernetics, Washington, USA).