Anti ki 67 antibody

Immunocytochemical staining was achieved via anti-Ki67 antibody (Sigma-Aldrich, Germany), anti-TGF beta antibody, and primary antibody mouse anti-human monoclonal antibody for iNOS (inducible nitric oxide synthase) from Lab Vision Laboratories (Thermo Fisher Scientific, USA). IHC was made on formalin-fixed, paraffin-embedded tissue according to the previous protocols [13 (link), 14 (link)]. All samples were blindly tested, and positive cells were randomly counted in 20 selected fields in both lamina propria and epithelial compartments using an Olympus microscope (Olympus, Japan).

Sixteen female SCID mice (Charles River „CB17/lcr-PrkdcSCID/lcrlcocrl”) were locally shaved and 3 × 10⁶ HUH-7 cells were injected subcutaneously into the flank of each mouse. Mice were divided into two groups and treated intraperitoneally with 0.2 mg/kg archazolid in 5% DMSO/10% solutol/PBS or equal amounts of 5% DMSO/10% solutol/PBS. Mice were treated daily. Measurement of tumors was done every 2 to 3 days with a caliper, using the formula a × b²/2. The average tumor volumes of the two groups were compared over time. IHC analysis of tumor tissue sections was performed as described previously [49 (link)] using anti-LAMP1-antibody (Abcam), filipin (Sigma Aldrich), anti-Ki67-antibody and haematoxylin (Sigma Aldrich). Animal experiments were approved by the District Government of Upper Bavaria in accordance with the German animal welfare and institutional guidelines.

Immunofluorescence assay of cultured cells was performed as previously reported [38 (link)]. Briefly, after transfection with calponin 2-siRNA, cells were incubated on chamber slides (Corning, USA). When the cells reached a sufficient number, they were fixed in 4% paraformaldehyde for 10 minutes. Goat serum blocking solution was used before overnight incubation with anti-Ki-67 antibody (Millipore, USA), after washes, Alexa Flour 488 labeled secondary antibody (Thermo Scientific, USA) was added for 30 minutes before 4, 6-diamidino-2-phenylindole (DAPI, Roche, Switzerland) stain of the nuclei. Confocal laser scanning microscopy was performed to acquire the images.

Four-micrometer sections of formalin-fixed and paraffin-embedded liver biopsy specimens were deparaffinized with xylene and rehydrated with decreasing concentrations of ethanol in water. Antigen retrieval was achieved in sodium citrate buffer (pH 6.0) for 20 min at 95 °C. Tissues were stained by immunohistochemistry with either anti-CD8a antibody (1:5000, Ebioscience, ThermoFisher Scientific, San Diego, CA, USA, clone 4SM15, catalog number: 14-0808-82), anti-CD4 (1:400, Cell signaling, Danvers, MA, USA, clone D7D22, catalog number: #25229), anti-CD3 (1:1000, Dako, Agilent, Santa Clara, CA, USA, clone A0452, catalog number: 790-4341), or anti-Ki67 antibody (1:150, Millipore, Merk, Darmstadt, Germany, catalog number: AB9260). The reaction was revealed using EnVision™+System-HRP (Dako, Agilent, catalog number: K4003) or DAB Substrate Kit (Abcam, Cambridge, UK, catalog number: ab64238), and sections were counterstained with hematoxylin. Images were acquired using a panoramic 250 Flash III digital slide scanner (3DHistech, Budapest, Hungary) or with AxioScan.Z1 (Carl Zeiss Microscopy GmbH, Jena, Germany) with a 20× objective and analyzed using Definiens System (Definiens AG, Munich, Germany) or ZEN2 blue edition software (Carl Zeiss Microscopy GmbH, Jena, Germany).