Cy3 affinipure goat anti rabbit igg h l

The NSCLC cell line A549 was plated on sheet glasses in a 6 mm plate. Cells on sheet glasses were fixed with 4% paraformaldehyde for 20 minutes and then washed three times with PBS. Cells were permeabilized with PBS containing 0.1% Triton X‐100 for 20 minutes and blocked in 5% bovine serum albumen for one hour. Sheet glasses were incubated with NDUFA4L2 and cytochrome C primary antibodies overnight at 4°C using the following dilutions: cytochrome C (mouse mAb, #12963, 1:200, Cell Signaling Technology) and NDUFA4L2 (rabbit polyclonal antibody, 1:25, Proteintech, Chicago, IL, USA). Sheet glasses were washed with tris‐buffered saline plus tween 20 three times and incubated with Cy3‐AffiniPure Goat Anti‐Rabbit IgG (H + L) and Alexa Fluor 488‐AffiniPure Goat Anti‐Mouse IgG (H + L) (Jackson ImmunoResearch, West Grove, PA, USA). Immunofluorescence was analyzed by fluorescence microscopy (Olympus BX51).

Prostate tissue samples were immediately fixed in ice-cold 4% formaldehyde supplemented with 1 tablet/10 ml of PhoSTOP (04 906 837 001; Roche). Prostate samples were embedded in paraffin, and 5-µm serial sections were cut. For histopathological analyses, paraffin sections were stained with H&E. For immunofluorescence staining, sections were processed as previously described (Ratnacaram et al., 2008 (link)), except that sections were incubated over night with primary antibodies diluted 1:200, unless indicated. Primary antibody used for immunofluorescence were directed against AKT pS473 (4060; Cell Signaling Technology), γH2AX (05-636; EMD Millipore; 1:600), p53 (CM5; Vector Labs), p53 pS15 (12571; Cell Signaling Technology), pHP1γ (2600; Cell Signaling Technology), RPA32 (GTX113004; Tebu-Bio), RPA32 pS4/S8 (A300-245A; Bethyl Laboratories), ATR (2790; Cell Signaling Technology), Ki67 (M7248; Dako), BrdU (600-401-C29; Rockland), ATM pS1966 (200-301-400; Rockland), Mdm2 (sc-965; Santa Cruz Biotechnology), Mdm2 pS166 (human)/pS163 (mouse; 3521; Cell Signaling Technology), PCNA (ab2426; AbCam), 53BP1 (NB100-305; Novus Biologicals). Secondary antibodies (CY3 AffiniPure goat anti–rabbit IgG [H+L], CY3 AffiniPure goat anti–mouse IgG [H+L], CY5 AffiniPure goat anti–mouse IgG [H+L], and CY5 AffiniPure donkey anti–rabbit IgG [H+L]) were from Jackson Immunoresearch.

After osteogenic induction and treatment, BMSCs were washed with PBS and then fixed in 4% paraformaldehyde for 15 min at room temperature. The cells were permeabilized with 0.1% Triton-X 100 in PBS for 10 min. After blocking with 5% BSA (Sigma-Aldrich; Merck KGaA) for 1 h at room temperature, the cells were incubated with primary antibodies against SIRT1 (Abcam; cat. no. ab110304, 1:2,000) and PCNA (Abcam; cat. no. ab92552, 1:500) at 4°C overnight. The cells were then incubated with DAPI, Alexa Fluor^® 488-AffiniPure goat anti-mouse IgG [H+L (Jackson ImmunoResearch Europe, Ltd.; cat. no. 115-545-003, 1:200] and Cy3- AffiniPure goat anti-rabbit IgG [H+L (Jackson ImmunoResearch Europe, Ltd.; cat. no. 111-165-003, 1:200)] at room temperature for 1 h. Images of cells were captured using a fluorescence microscope (Olympus Corporation; magnification, ×1,000).