Anti ki67 ab15580

Bombyx mori Cocoons were purchased from Huzhou Xintiansi Biotech Co., Ltd (Huzhou, China). Biliverdin (Catalog No. 50-981-690) was bought from Thermo Fisher Scientific (Waltham, MA, US). Lithium bromide, Na₂CO₃, and fluorescein isothiocyanate (FITC) were from Sigma-Aldrich (St Louis, MO). Indocyanine green (ICG) (Catalog No. MB4594) was obtained from Meilunbio VR (Dalian, China). Fetal bovine serum (FBS), trypsin, phosphate buffered saline (PBS) and Dulbecco's Modified Eagle's Medium (DMEM) were purchased from Life Technologies (Grand Island, NY). The cell lines, including C6, U87, GB261 and RAW 264.7, were obtained from the Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences (Shanghai, China). The antibodies of Anti-Ki67 (ab15580,), anti-α-SMA (ab52575), and anti-IL-1 (ab9722) were obtained from Abcam (Abcam, Cambridge, MA, US). Anti-TNF-α (52b82) antibody was bought from Santa Cruz Biotechnology (Dallas, TX, US).

SiHa cells were seeded on coverslips and exposed to treatments. Cells were fixed with 4% paraformaldehyde for 30 min and permeabilized with 0.1% Triton X for 5 min. Blocking was performed with 3% bovine serum albumin for 20 min. Cells were incubated with antibodies against responding primary antibody (anti-Ki67, ab15580; Abcam) and linked to Alexa Fluor^® 488 or 555 (Cell Signaling Technology). After being washed, the nuclei were counterstained with 4′-6-diamidino-2-phenylindole (DAPI; Sigma-Aldrich, St. Louis, MO, USA). The coverslips were mounted using Vectashield mounting medium (Vector Laboratories, Burlingame, CA, USA). Immunofluorescence was visualized using an Olympus BX41 microscope (Tokyo, Japan).

Tissue sections on the slides were deparaffinized in xylene, rehydrated in ethanol, and rinsed in double-distilled water. Antigen retrieval was at 92C for 20 minutes using LabVision™ HIER Buffer L (Thermo Fisher Scientific Inc), slides were rinsed in 1xPBS, and tissue sections on the slides were incubated in normal goat serum from Vector Laboratories for 2 hours at room temp. Primary antibody from Abcam® (Anti-Ki67 ab15580) or (anti-alpha smooth muscle actin ab7817 and vimentin ab8069) or Santa Cruz biotechnology®, Inc. (HMG-1 sc-26351) was then added overnight at 4°C. On the following day, slides were rinsed in 1xPBS and the secondary antibody (Alexa Fluor® 594-conjugated AffiniPure Goat Anti-Rabbit for Ki67) or (Goat anti-mouse IgG1, Alexa Fluor® 594 for vimentin with Alexa Fluor® 488-conjugated AffiniPure goat anti-rabbit for alpha actin) or (Alexa Fluor® 594-conjugated AffiniPure Donkey anti-goat for HMG-1) was incubated on the slides for 2 hours at room temp. Slides were then rinsed in 1xPBS and Vector laboratories DAPI mounting medium was added to each slide. Tissue sections were viewed with an Olympus BX-51 epi-fluorescent microscope and images were photographed with an Olympus DP71 camera. These images were captured using Olympus cellSens Standard 1.11 software (Waltham, MA).

Primary antibodies to β-actin (SC-47778, 1:1000), CaMKK-ß (SC-50341, 1:1000), and AMPKα1/2 (SC-25792, 1:1000) were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Primary antibodies to p-CaMKKβ (12818S, 1:500), mTOR (2983S, 1:1000), S6 ribosomal protein (2217S, 1:1000), and p-S6 ribosomal protein (2211S, 1:1000) were purchased from Cell Signaling (Danvers, MA). Anti-p-AMPKα1/2 (11183, 1:1000) and anti-p16 (41296, 1:1000) antibodies were from Signalway (College Park, MD, USA). Anti-LC3B (NB100-2220, 1:500) and anti-p21 (NBP2-29463, 1:500) antibodies were purchased from Novus (St. Louis, MO, USA). Anti-p-mTOR (ab63552) and anti-Ki-67 (ab15580) antibodies were purchased from Abcam (Cambridge, UK). Anti-mouse (31430), anti-rabbit (31460), anti-goat (31402), and Alexa Fluor 488 (A11008) immunoglobin G secondary antibodies (1:5000) were purchased from Invitrogen (Carlsbad, CA, USA). The anti-OR2H2 primary antibody (1:1000) was purchased from Antikoerper-online.de (Aachen, Germany). L-Cis diltiazem was from Abcam (Cambridge, UK). SQ22536 and thapsigargin were purchased from Enzo Life Science (Farmingdale, NY, USA) and U73122 was from Sigma-Aldrich (St. Louis, MO, USA). Aldehyde 13-13 was obtained from Henkel (Düsseldorf, Germany). A769662, an AMPK agonist, was purchased from Cayman (Ann Arbor, MI, USA). Rapamycin was obtained from LC Laboratories (Woburn, MA, USA).