Dmrb fluorescence microscope

Ki67 staining (proliferation) and active-caspase3 staining (apoptosis) were performed on 4 liver sections per tumor. Those areas were randomly selected from the slides based on the DAPI staining, to compensate for the possibly uneven distribution of the apoptotic/proliferating cells within the tumor mass. Rehydrated sections were heated for 20 minutes in Target Retrieval Solution (pH 6.1, Dako). After blocking with 20% normal goat serum (Dako) in PBS, slides were incubated overnight at 4°C with polyclonal rabbit anti-Ki67 antibody (1 : 500, Abcam, ab15580) or rabbit anti-active caspase3 antibody (1 : 400, Cell Signaling Technology, 9664S) each diluted in Dako Antibody Diluent. As secondary antibody the Alexa fluor 488 goat anti-rabbit antibody was used for 1 hour at room temperature and nuclei were stained with DAPI (dilution 1/10000, Dako). Images were taken using the Leica DMRB Fluorescence microscope. Ki67, active caspase 3 and DAPI positive cells were counted using the cell counter plugin of the Image J software (National Institute of Health (NIH), Bethesda, USA). At least 2000 cells were counted per liver. The proliferation and apoptosis index was calculated by counting the number of Ki67 or active caspase 3 positive cells, respectively, divided by the number of DAPI positive nuclei (total number of cells).

Both PC3^control and PC3^AGR−2sh cells were grown in culture until 50% confluence in chambered slides, washed thoroughly in PBS and fixed in ice-cold 3.7% paraformaldehyde in PBS containing 0.1% Triton-X-100, for 20 min, at room-temperature. Cells were washed thoroughly and incubated overnight at 4°C in rat monoclonal human AGR-2 antibody. Following PBS wash, cells were incubated with alexa-fluor-594 labeled anti-rat IgG (Molecular Probes, Eugene, OR), for 30 minutes, at room temperature. Cells were washed and nuclei were stained with DAPI for contrast. The fluorescent labeled cells were mounted using Vectashield (Vector Labs, Burlingame, CA) and viewed in a Leica DMRB fluorescence microscope.

PFA fixed, 30-μm-thick coronal brain slices were obtained and processed for immunofluorescence analysis. Immunofluorescence was performed, after blockade with 5% goat serum, by overnight incubation at 4°C with a GluA1 CTD primary antibody (rabbit, Synaptic Systems, #182–003) followed by incubation with an Alexa 488 anti-rabbit secondary antibody (Invitrogen). Images were obtained using a Leica DMRB fluorescence microscope and processed with ImageJ.

Dissected pancreata were fixed in Bouin’s solution, embedded in paraffin, and cut into 5-μm sections. Insulin (mouse monoclonal, Sigma-Aldrich) and glucagon (rabbit polyclonal, Abcam) immunostaining and morphometric analysis were performed blindly as previously described using a DMRB Fluorescence microscope (Leica) (46 (link)). Dispersed islets and INS1E cells grown overnight on poly-l-lysine coverslips were fixed in 4% PFA, permeabilized, and immunostained with primary (listed in the Reagents section) and secondary antibodies (Alexa Fluor 488 and 594, Invitrogen). Cells were mounted on glass slides and imaged using a TCS SP5 II Confocal microscope (Leica).

To determine whether the binding of ¹⁸F-fluoride in regions of Von Kossa and Alizarin Red S was specific for hydroxyapatite deposition, categorisation of these regions using a fluorescein-bisphosphonate probe was undertaken. Fluorescein-bisphosphonate is a highly sensitive and specific probe for identifying regions of microcrystalline hydroxyapatite. Incubation and binding in tissue has previously been described in detail^{13 (link)}. Briefly, sections were de-waxed in xylene and incubated with fluorescein-bisphosphonate (1 µM) for 2 h, washed in water (2 ×) followed by incubation with 2% Alizarin Red S (250 µL) for 5 min. Sections were washed in water (3 ×) and subsequently incubated with 4′,6-diamidino-2-phenylindole (500 nM) for 5 min. Sections were washed with water (1 ×) and then mounted using ProLong Gold Antifade. Fluorescence signal was detected under a Leica DMRB fluorescence microscope.

The cells were seeded into 24-well plates at a density of 5 × 10⁴ per well. Twenty-four hours later, the cells were treated with 1.4 ppm AgNPs w and w/o 1 mM α-lipoic acid or were left untreated in the control. Twenty-four hours later, the cell culture medium was removed, and the cells were stained according to the instructions of the Mitochondrial Staining Kit—Red Fluorescence—Cytopainter (Abcam, Berlin, Germany). Randomly chosen fields were examined at 400× magnification using a Leica DMRB fluorescence microscope. Images were captured using a SPOTTM FLEX 15.2 64 Mp shifting pixel digital color camera (Diagnostic Instruments, Inc., Sterling Heights, MI, USA) and analyzed with SPOT Advanced 4.6 software (SPOT Imaging^TM, Sterling Heights, MI, USA).