Ki 67 clone sp6

For immunohistological analysis, explants attached to Millicell‐CM culture plate inserts were fixed overnight with 4% paraformaldehyde, embedded in paraffin, and sectioned at a thickness of 3 μm. Sections were stained with rabbit polyclonal antibodies to cleaved caspase 3 (Cell Signaling Technology, Danvers, MA), to Ki67 (clone SP‐6, Thermo Fisher Scientific), and to γH2AX (phospho‐S139; Abcam, Cambridge, UK) or with a mouse monoclonal antibody to phosphorylated histone H3 (Cell Signaling Technology). Immune complexes were detected as described previously 18. Quantifications were performed with ImageJ.

The tissue was processed for paraffin embedding. Paraffin sections were cut at a thickness of 6 μm. Tissues were stained for histological analysis with haematoxylin and eosin. Primary antibodies used were HNF4α (SC‐6556, Santa Cruz), YAP1 (#4912, Cell Signaling) and Ki67 (clone SP6, RM‐9106‐R7 Thermo Scientific) followed by secondary antibodies from Reactolab using Vectastain Elite ABC Kit for goat IgG (PK‐6105) or rabbit IgG (PK‐6101). For quantification of Ki67‐positive hepatocytes in the knockdown experiment without PH, 20 high power fields were photographed per sample and hepatocytes were manually counted for HNF4α‐ and Ki67‐positive reactions. For the quantification of the number of hepatocytes in proliferation in aged and young mice, the 3DHISTECH Pannoramic MIDI Scanner and 3DHISTECH Quant 2.0 software were used. A proliferation index (p.i.) was calculated as a ratio of Ki67‐positive nuclei to total number of hepatocytes counted. The results are presented as the p.i. ± SD and were compared with an unpaired Student's t‐test.

Three days after the second delivery, three mice from each experimental group were sacrificed and tumors were excised and fixed in IHC zinc fixative (BD Pharmingen, BD Biosciences, San Jose, CA, USA) overnight and afterwards embedded in paraffin. Three consecutive 5-μm thick sections were cut from each paraffin block. The first section of each tumor sample was stained with hematoxylin and eosin (HE) for the determination of necrosis. The two remaining sections were immunohistochemically stained with rabbit monoclonal antibodies against Ki-67 (clone SP6, Thermo Fisher Scientific) at dilution 1:1200 for determination of proliferation of tumor cells or rabbit monoclonal antibodies against Cleaved Caspase-3 (Casp-3, Cell Signalling Technology, Danvers, MA, USA) at dilution 1:1500 for determination of apoptosis. As the colorogenic reagent a peroxidase-conjugated streptavidin–biotin system (Rabbit specific HRP/DAB detection IHC kit ab64261, Abcam, Cambridge, UK) was used followed by hematoxylin counterstaining. Three different fields of immunohistochemically stained sections were captured with a DP72 CCD camera (Olympus, Hamburg, Germany) connected to a BX-51 microscope (Olympus). Three independent observers blinded to the treatment groups quantified the percentage of tumor necrosis and the number of Ki-67- or Casp-3-positive cells on the acquired images.

Expression of JNK1 and -2 was determined in specimens of patients with pancreatic ductal adenocarcinoma, who underwent tumor resection at our department. Specific recombinant rabbit monoclonal antibodies were used for JNK1 (ab110724, Abcam) and JNK2 (ab76125, Abcam). Immunohistochemistry was performed using formalin-fixed and paraffin-embedded 5 µm sections in dilution 1:250 in combination with a secondary biotinylated goat anti-rabbit antibody and a Vectastain Elite ABC kit (Vector Lab, Burlingame, CA) according to the protocol of the manufacturer. For immunohistochemical analysis of orthotopic tumors, the following antibodies were used according to the protocols of the manufacturers: Ki67 (clone SP6, Thermo Fischer Scientific, Fremont, CA), p-c-Jun (54B3, Cell Signaling Technologies), MMP-2 (SAB2108458, Sigma-Aldrich, St. Louis, MO), MMP-9 (ITA1401, G-Biosciences, St. Louis, MO), Vimentin (D21H3, Cell Signaling Technologies). All samples were collected immediately during the operation with prior obtained informed consent from all patients.

For histology, mice were intracardially perfused with 20 ml cold PBS and fixed by perfusion with 10 ml of 4 % paraformaldehyde (PFA). Spinal cords were removed and kept in PFA for 48 h at 4 °C. The fixed spinal cords were cut into 3 mm thick transverse segments and embedded in paraffin. To evaluate demyelination, spinal cord sections were stained with Luxol Fast Blue and subsequently incubated with Periodic acid-Schiff. Immunohistochemistry was performed using the biotin-streptavidin peroxidase technique (K5001, Dako) in an immunostainer (AutostainerLink 48, Dako). Sections were pretreated in a steamer (treatment solutions pH 6.0 or pH 9.0 (Dako)) before incubation with the primary antibodies against CD3 (clone CD3-12, BioRad, 1:100) or Mac3 (clone M3/84, BD, 1:100) or B220 (clone RA3-6B2, BD, 1:200). DAB (3,3ʼ-Diaminobenzidin) was used as a chromogen. For B220/Ki67 double-immunofluorescence staining, B220 (clone RA3-6B2, BD, 1:100) and Ki67 (clone SP6, Thermo Scientific, 1:100) were used as primary antibodies; AF488- and AF594-labeled secondary antibodies (both 1:100) were used for visualization. Stained sections were analysed with a keyence microscope and pictures were taken with an Axioplot camera. ImageJ v1.48 was used to manually count infiltrated cells and measure areas.