Axiovision 3

The isolation of bone marrow precursors and the in vitro osteoclastogenesis experiments were performed as described previously^{19 (link)}. In brief, bone marrow cells were isolated from the femurs of 4–6 week-old C57BL/6 male mice and incubated in α-MEM containing 10% fetal bovine serum and M-CSF (10 ng/ml). After 24 h, the nonadherent cells were harvested and cultured in α-MEM containing 10% fetal bovine serum and M-CSF (20 ng/ml) for 3 days. After washing out the nonadherent cells, the adherent cells were used as bone marrow-derived monocytes/macrophages (BMMs). For osteoclast formation, isolated BMMs were further incubated with 200 ng/ml RANKL and 30 ng/ml M-CSF. After 5 days, these cells were fixed and stained for TRAP using a TRAP staining kit (Sigma-Aldrich). The number of pink-colored TRAP-positive (TRAP+) multinucleated cells (MNCs; >3 nuclei) or large TRAP+ MNCs with >20 nuclei were counted and are presented as relative percentages. The cells were observed using a Zeiss Axiovert 200 microscope, and images were obtained with an AxioCam HR microscope camera (Carl Zeiss, Göttingen, Germany) equipped with Axio Vision 3.1 software (Carl Zeiss). RAW264.7 cells were cultured in DMEM containing 10% fetal bovine serum.

NeuO is a membrane-permeable fluorescent probe that selectively stains live neurons. The hippocampal cells were cultured on glass coverslips. The neurons were subjected to DIM and ischemia and then incubated with 0.125 µM NeuO suspended in Neurobasal for 2 h. Then, the labeling medium was replaced with fresh medium, and the cells were incubated for another 2 h. Fluorescence images were recorded and developed using an inverted fluorescence microscope (AxioObserver, Carl Zeiss) with an excitation wavelength of 350 nm (UV fluorescence) and Axiovision 3.1 software (Carl Zeiss, Germany).

In a separate set of mice (n = 5), the kidneys of the mice were extracted following sacrifice and immediately processed to evaluate the DNA damage as per the standard Comet assay protocol [29 (link),30 (link)]. The DNA damage was assessed by measuring the length of DNA migration, which included the diameter of the nucleus and the migrated DNA, using image analysis Axiovision 3.1 software by Carl Zeiss (White Plains, NY, USA).

Through the canonical NF-κB signaling pathway, IKK phosphorylates IκB proteins, thus allowing the release of free p50/p65 NF-κB dimers that translocate to the nucleus. For immunocytochemical experiments, cells were cultured and treated on sterile glass coverslips placed in a 24-well microplate. Cells were washed twice with PBS and fixed with 4% paraformaldehyde for 40 min at room temperature (RT). After washes with PBS, 0.5% Triton X-100 (v/v in PBS) was added for 5 min at 4°C and then washed with PBS. Cells were incubated with blocking buffer (1% BSA in PBS) for 45 min prior to incubation for 1 h at RT with primary antibody anti-p65, diluted 1 : 100 in PBS with 0.5% BSA. Cells were washed and incubated for 45 min with FITC-conjugated secondary antibody diluted in PBS containing 0.5% BSA. Cells were washed and incubated for 10 min in DAPI diluted 1 : 10.000 in PBS. Finally, the coverslips were mounted on glass slides using DABCO. Images were acquired on a Carl Zeiss Axioplan 2 imaging microscope using an AxioCam HR CCD camera and AxioVision 3.1 software (Carl Zeiss, Göttingen, Germany).

C4-2 tumors were isolated from mice at the end of the study described above and were prepared for immunohistochemical assessment of apoptosis and Ki-67. C4-2 tumors were sectioned and stained with hematoxylin and eosin (HE) and the desired areas marked along with their corresponding paraffin blocks. The rabbit anti-human anti-Cleaved Caspase 3 (Asp175) (5A1E) (1:50; Cell Signaling Technology, Danvers, MA, USA), rabbit anti-human anti-Ki 67 proliferating markers (1:50; Cell Signaling Technology, Danvers, MA) and rabbit polyclonal anti-p21 (1:150; Santa Cruz Biotechnology Inc) were used for immunohistochemical staining. All sections used for immunohistochemistry were lightly counterstained with 5% (w/v) Harris hematoxylin. Five fields of each slide were randomly chosen and images taken (400), using an AxioCam HR CCD mounted on an Axioplan 2 microscope and Axiovision 3.1 software (Carl Zeiss, Canada). Positively stained cells and whole cells in each image were counted and the percentage of positive cells was calculated. The TMAs were manually constructed (Beecher Instruments, MD, USA) by punching quadruplicate cores of 1 mm for each sample giving a total of 144 cores. All scoring was done blinded with respect to treatment by LF and based on relative immunoreactive intensity on a four-point scale.

For BrdU incorporation assay, cells were labeled with 10 μm BrdU (Roche, Indianapolis, IN, USA) for 2 h, fixed with 4% para-formaldehyde, and immunostained with anti-BrdU antibody (Roche) followed by staining with Cy™3-conjugated goat anti-mouse IgG (115-165-146; Jackson ImmunoResearch Laboratories, West Grove, PA, USA) and counter-stained with DAPI. BrdU-positive cells were scored under a fluorescent microscope and presented as the percentage of BrdU-positive nuclei over total number of nuclei counted. At least 300 nuclei were counted. For immunofluorescence, cells were fixed with 4% paraformaldehyde, immunostained with primary and secondary antibodies in 4% BSA, and counter-stained with DAPI. Antibodies used include anti-p53 (DO-1; Santa Cruz Biotechnology, Santa Cruz, CA, USA) and goat anti-mouse Alexa Fluor 488 (A11001; Santa Cruz Biotechnology). Cell images were recorded with an Axiovert 200M microscope (Carl Zeiss, Oberkochen, Germany) and analyzed with axiovision 3.1 software (Carl Zeiss).