Axioimager m1 fluorescent microscope

Histology was performed on 10μm neutral buffered formalin-fixed cryosections of proximal tibial epiphyses from two week old wildtype, ColX^N617K, Xbp1^CartΔEx2, or C/X mice. Toluidine blue staining [12 (link)] and immunofluorescent analyses using antibodies specific for collagen II or collagen X [14 (link)] were performed as described. Immunofluorescent analysis of ATF4 expression was performed using 1:100 rabbit anti-human ATF4 antibody (D4B8; Cell Signaling Technology) and an appropriate fluorescent secondary antibody (10μg/ml; Molecular Probes, Life Technologies), as follows. Prior to antigen retrieval, all sections were incubated for 10 min at room temperature in PBS, 0.2% Triton X-100 (Sigma-Aldrich). For ATF4 antigen retrieval, sections were incubated for 10 min at room temperature in 1% SDS (Sigma Aldrich) in PBS. All immunofluorescence sections were counterstained and mounted using VECTASHIELD Mounting Medium with DAPI (Vector Laboratories, Inc), and visualized by fluorescent microscopy with an Axio Imager M1 fluorescent microscope (Zeiss).

The following probes were used: pTa71 (18S-28S rDNA) [52] (link), pCT4.2 (5S rDNA) [53] (link), the Arabidopsis-type telomere probe (5'-CCCTAAA-3')₃ synthesized with a TAMRA label (ZAO “Syntol”, Moscow, Russia) and the CS-1 probe (C.sativa subtelomeric repeat (JX402748)). All DNA probes were labeled by nick translation and PCR according to the manufacturer’s instructions (Boehringer, Germany) with biotin-16-dUTP (CS-1, pCT4.2) or digoxigenin-11-dUTP (pTa71). FISH experiments were performed as described by Karlov et al. [21] . The chromosomes were counterstained with 1 mg/ml DAPI and mounted in Vectashild (Vector laboratories, UK). An AxioImager M1 fluorescent microscope (Zeiss) was used to observe chromosome preparations. The metaphase plates with fluorescent signals were photographed with a monochrome AxioCam MRm CCD camera and visualized using Axiovision software (Zeiss). Metaphase chromosomes were classified according to Levan et al. [54] based on their arm ratio and FISH hybridization pattern. In each experiment, at least 20 mitotic metaphase plates from each of the 11 male and 8 female plants were analyzed.

THP-1 monocytes were applied to 24-well transwell migration plates (Invitrogen, USA), consisting of upper and lower chambers, separated by membranes punctuated with pores of 5 μm in diameter. The 1 × 10⁵ monocytes were seeded in a final volume of 150-μl basal medium into the upper chamber. Conditioned medium was prepared from confluent EC treated with 15d-PGJ₂ or MG132 for 18 h; the cells were washed, fresh medium was added, and then the cells were treated with TNF- α (0.2 ng/ml for 6 h). After incubation, conditioned medium was cleared by centrifugation (14,000 × g for 10 min at 4°C). Conditioned medium was added to the lower chamber in a final volume of 500 μl as a chemoattractant. Plates were incubated for 2 h at 37°C. Membranes were washed with PBS and were placed into 4% formaldehyde for 15 min to fix cells adherent to the underside of the membrane. Membranes were washed with PBS, and the upper chambers immersed in DAPI solution for 3 min, for nucleic acid staining. Following two PBS washes, the adherent cells were visualized using a Zeiss AxioImager M1 fluorescent microscope. The number of cells in five random 20× fields was counted, and the average value was expressed as a percentage of control.

For static adhesion assay, 4 × 10⁴ EC were seeded in a 96 well plate for 24 h and then treated with hydrolysates for 18 h. Subsequently, EC were stimulated with TNF-α (0.5 ng/ml) for 6 h. After treatments, the wells were washed three times with medium and 1 × 10⁵ fluorescein-labelled THP-1 monocytes were added to each well and incubated for 30 min at 37°C. After incubation, the wells were washed three times with medium and adherent monocytes were measured in a Spectramax M2 (Molecular devices, CA) plate fluorescence reader with 485 nm excitation and 530 nm emission wavelength. For photomicrographs, fluorescence-labelled adherent monocytes in the 96-well plate were analyzed using a Zeiss AxioImager M1 fluorescent microscope. Adhesion assay experiments were repeated in triplicate and the average value was expressed as a percentage of control (vehicle).

Cells were fixed with 4% paraformaldehyde (PFA) at 4 °C, permeabilized with 0.2% Triton X-100 at 4 °C and blocked in BSA 1% dissolved in phosphate-buffered saline (PBS) (w/v) at 37 °C. Samples were then co-immunostained overnight (ON) at 4 °C, using a rabbit polyclonal telomeric protein TRF1 antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA) in combination with a mouse monoclonal anti-53BP1 antibody (Millipore Corp. 290 Concord Rd, Billerica, MA, USA). After washes in PBS/BSA 1% samples were incubated with the secondary antibody (anti-mouse Alexa 546 and anti-rabbit Alexa 488, Invitrogen, Life Technologies, Carlsbad, CA, USA) for 1 h at 37 °C. Finally, slides were washed in PBS/BSA 1%, counterstained with DAPI (Sigma-Aldrich), and images were acquired with an Axio-Imager.M1 fluorescent microscope (Carl Zeiss, Jena, Germany) and analyzed using ISIS software (Metasystems, Milano, Italy). The frequency of DNA damage marker foci and TRF1/53BP1 colocalization dots per cell were scored in 100 nuclei in three independent experiments.

After irradiation and/or hyperthermic treatment a portion of the BxPC3 cells were seeded in slide flasks (Thermo Fischer Scientific, Waltham, MA, USA) with 3 mL fresh medium and collected 6 and 24 h later. Cells were fixed in 4% paraformaldehyde (PFA), permeabilized with 0.2% Triton X–100 and blocked at 37 °C in BSA 1% (w/v) dissolved in phosphate-buffered saline (PBS). Samples were then coimmunostained overnight (ON) at 4 °C, using a rabbit polyclonal anti–53BP1 antibody (Novus Biologicals, Littleton, CO, USA) in combination with a mouse monoclonal anti–γH2AX antibody (Millipore, Temecula, CA, USA). After washes in PBS/BSA 1% samples were incubated for 1 h at 37 °C in the secondary Alexa 546 anti-mouse and Alexa 488 anti-rabbit antibodies (Invitrogen, Life Technologies, Carlsbad, CA, USA). Finally, slides were washed in PBS/BSA 1%, counterstained with DAPI (Sigma-Aldrich, St. Louis, MO, USA) and mounted using the fluorescent mounting medium Vectashiled (Vector Laboratories, Burlingame, CA, USA). Images were acquired with an Axio-Imager.M1 fluorescent microscope (Zeiss, Jena, Germany) and analyzed using ISIS software (Metasystems, Milano, Italy). The frequency of both the DNA damage marker foci per cell were scored in 100 nuclei in four independent experiments.

For static adhesion assay, 4 × 10⁴ EC were seeded in a 96-well plate for 24 h and then treated with 15d-PGJ₂ or MG132, for 18 h. Subsequently, EC were stimulated with TNF-α (0.2 ng/ml) for 6 h. After treatments, the wells were washed three times with medium, and 1 × 10⁵ fluorescein-labeled THP-1 monocytes were added to each well and incubated for 30 min at 37°C. After incubation, the wells were washed three times with fresh medium and adherent monocytes were measured in a Spectramax M2 (Molecular Devices, CA, USA) plate fluorescence reader with 485-nm excitation and 530-nm emission wavelength. For photomicrographs, fluorescence-labeled adherent monocytes in the 96-well plate were analyzed using a Zeiss AxioImager M1 fluorescent microscope. Adhesion assay experiments were repeated in triplicate, and the average value was expressed as a percentage of vehicle control.