Rabbit anti ki67

The following primary antibodies were employed for Western blotting: mouse anti-β-tubulin (1:1000, cod. T5201; Sigma-Aldrich Corp.), mouse anti-β-actin (1:2000, cod. A5441; Sigma-Aldrich Corp.), rabbit anti-GAPDH (1:1000, cod. G9545, Sigma-Aldrich Corp.), mouse anti-cathepsin D (1:100, cod. IM03; Calbiochem, St. Louis, MO, USA), mouse anti-histone H3 (1:500, cod. 61475; Active Motif, Carlsbad, CA, USA). The secondary antibodies used for Western blot analysis were the following: horseradish peroxidase-conjugated goat anti-mouse IgG (1:10,000, cod. 170-6516; Bio-Rad, Hercules, CA, USA) and horseradish peroxidase-conjugated goat anti-rabbit IgG (1:10,000, cod. 170-6515: Bio-Rad, Hercules, CA, USA). The primary antibodies employed for immunofluorescence staining are listed below: mouse anti-cathepsin D (1:100, cod. IM03; Calbiochem), rabbit anti-cathepsin D (1:100; EMD Biosciences, Calbiochem, San Diego, CA, USA), rabbit anti-p27 (1:100, cod. 2552; Cell Signaling, Danvers, MA, USA), rabbit anti-Ki-67 (1:100, cod. HPA001164; Sigma-Aldrich), mouse anti-E-cadherin (1:50, cod. 14472S; Cell Signaling) and rabbit anti-N-cadherin (1:50, cod. 4061S; Cell Signaling). The secondary antibodies were goat-anti rabbit IgG Alexa Fluor Plus 488 (1:1000, cod. A32731; Invitrogen, Waltham, MA, USA) and goat-anti mouse IgG Alexa Fluor Plus 555 (1:1000, cod. A32727; Invitrogen).

The following primary antibodies were employed for either immunofluorescence or western blotting: mouse anti-ARH-I (for IF 1:250, for WB 1:1000; cod. ab45768; Abcam, Cambridge, UK), mouse anti-p21 (for IF 1:100, for WB 1:200; cod. sc-817; Santa Cruz Biotechnology, Dallas, TX, USA), rabbit anti-LC3 (1:2000; cod. L7543; Sigma-Aldrich), rabbit anti-p62 (1:500; cod. 8025; Cell Signaling, Danvers, MA, USA), mouse anti-β-actin (1:2000; cod. A5441; Sigma-Aldrich), mouse anti-BECLIN-1 (1:500; cod. 612112; BD Biosciences, Franklin Lakes, NJ, USA), goat anti-BECLIN-1 (1:250; cod. sc-10086; Santa Cruz Biotechnology), rabbit anti-VPS34 (1:500; cod. 4263; Cell signaling), mouse anti-STAT3 (1:500; cod. 9139; Cell Signaling), rabbit anti-phospho (Tyr705) STAT3 (1:1000; cod. 9145; Cell Signaling), rabbit anti-Ki-67 (1:100; cod. HPA001164; Sigma-Aldrich), mouse anti-BCL-2 (1:500; cod. 15071; Cell Signaling), mouse anti-IL-6R (1:1000; cod. AHR0061; Invitrogen, Waltham, MA, USA), rabbit anti-cyclin D1 (1:500; cod. 2978; Cell Signaling), rabbit anti-p38 (1:500; cod. 9212; Cell Signaling), rabbit anti-GAPDH (1:1000, cod. G9545, Sigma Aldrich), and mouse anti-ERK1/2 (1:500; cod. 05-1152; Millipore, Burlington, MA, USA).

Monolayer-cultured rBM-NCPs were fixed with 4% (wt/vol) paraformaldehyde, blocked, and then incubated with primary antibodies, including rabbit-anti-CD133 (1:200, Abcam), rabbit-anti-p75 (1:1000, Cell Signaling), rabbit-anti-nestin (1:50, Sigma), mouse-anti-vimentin (1:25, Sigma), mouse-anti-CD29 (1:100, Sigma), and rabbit-anti-Ki67 (1:200, Sigma), overnight at 4 °C, followed by reaction with FITC-anti-rabbit-IgM (Sigma), Cy3-anti-rabbit-IgM (Santa Cruz), TRITC-anti-mouse-IgM (Santa Cruz) or FITC-anti-mouse-IgM (Sigma), and Hoechst 33342 (Sigma) counterstaining. The cell samples were observed under a confocal laser scanning microscope (TCS SP2, Leica Microsystems, Germany).
Induced Schwann cells were subjected to immunofluorescent staining with mouse anti-S100β (1:250, Sigma), rabbit-anti-glial fibrillary acidic protein (anti-GFAP, 1:200, DakoCytomation), and rabbit-anti-p75 (1:1000, Cell Signaling Technology) respectively, followed by the same reaction with the second antibody and Hoechst 33342 counterstaining.

The Ki67 staining was performed as previously described.⁽⁴⁷⁾ Briefly, the primary osteoblasts isolated from long bones were plated on 0.32 cm² of each well in a 96‐well plate (6.4 × 10³ cells/well). After 48 hours of seeding, the culture medium was replaced with osteogenic induction medium. At day 17, the cells were fixed with 4% paraformaldehyde for 10 minutes, permeabilized with 0.2% Triton X‐100 (cat. X100; Sigma) for 10 minutes, blocked with 30 μL of IHC select blocking buffer (cat. 20773; Merck) for 30 minutes, and stained with rabbit anti‐Ki67 (cat. Ab15580; Abcam, Cambridge, UK) antibody (1:100 in blocking buffer) (1 hour; RT). The cells were washed thrice in PBS and 30 μL of Alexa‐488 anti‐rabbit secondary antibody (1:400 in blocking buffer) (cat. A21206; Thermo Fisher Scientific) was added (45 minutes; RT). The cells were washed again thrice with PBS followed by DAPI staining (1 μg/mL) (cat. 62248; Thermo Fisher Scientific) (5 minutes; RT). The samples were imaged using ImageXpressMicro confocal microscope (Molecular Devices, San Jose, CA, USA)⁽⁴⁷⁾ and were analyzed using cell profiler software.⁽⁵⁵⁾

Tumor sections (5-μm thick) were immunostained to quantify adenovirus and cleaved caspase 3. In brief, appropriate primary antibodies [goat anti-cleaved caspase 3 (diluted 1:200, Cell Signaling Technologies), rabbit anti-adenovirus (diluted 1:3000, Abcam), or rabbit anti-Ki67 (diluted 1:200, Merck Millipore)] were incubated with sections at 4ºC overnight. The HRP-labeled secondary antibodies [goat anti-mouse IgG/HRP (ZSGB-BIO) or goat anti-rabbit IgG/HRP (ZSGB-BIO)] for immunochemical staining were developed via a peroxidase reaction with diaminobenzidine as the chromogen.

Immunofluorescence staining was performed as we previously described^{8 (link)}. Briefly, after washing twice with PBS, HepG2 cells were fixed with 4% paraformaldehyde in PBS for 20 min at room temperature, at indicated times. Then, cells permeabilization with 0,1% Triton X-100 twice for 10 min, and blocking with 2% BSA were performed. Cells were incubated overnight with primary antibodies, including rabbit anti-p21 (1:100, Santa Cruz), rabbit anti-Ki-67 (1:100 Millipore) and mouse anti-Mdm-2 (1:100, Santa Cruz). Secondary antibodies Alexa Fluor 488-conjugated goat anti-rabbit IgG or Alexa Fluor 488-conjugated goat anti-mouse IgG (1:100, Thermo Fisher Scientific) were used according to the manufacturer’s instructions. The samples were incubated at room temperature for 1 h and washed 3 times with PBS. Nuclei were counterstained with DAPI, and samples were mounting with prolong Gold antifading solution (Thermoscientific). Primary antibody was omitted in negative controls (data not shown). Cells were visualized and photomicrographed under an inverted fluorescence microscope (Nikon). Positive Alexa 488 cells were analyzed and quantified using FIJI-Image J software (Bethesda, MD, USA).