Axiovert 40 cfl microscope

Visualization of actin ring, VNR and CTR expression was analysed by immunofluorescence microscopy. CD14+ monocytes were seeded on glass cover-slips in 24-well plates and exposed to LCPUFAs (40 μM) or vehicle control in the presence of differentiation factors. At the end of culture cells were stained for actin, VNR and CTR as described previously [33 (link)]. Briefly, cells were washed twice with PBS and fixed with 3.7% (v/v) formaldehyde in PBS for 15 min. Cells were permeabilised for 5 min with 0.1% Triton X-100 and stained for actin with 5 U/ml Alexa Fluor 568-Phalloidin and 35 μg/ml Hoechst for nucleus. For VNR or CTR staining, 50 μg/ml mouse anti-VNR or anti-CTR antibodies were employed followed by staining with 2 μg/ml Alexa Fluor 488-goat anti-mouse secondary antibodies. Images were acquired by confocal laser scanning microscopy using a Zeiss Axiocam MRc5 camera attached to a Zeiss Axiovert 40 CFL microscope (Carl Zeiss AG, Oberkochen, Germany) using the appropriate filter sets: Hoechst (Excitation: 352 nm, Emission: 455 nm); Alexa Fluor 488 (Excitation: 490 nm, Emission: 525 nm) Alexa Fluor 568-Phalloidin (Excitation: 578 nm, Emission: 600 nm).

Cell death in MIN-6 cells was evaluated using a Cell Death ELISA^PLUS, detecting enrichment of cytoplasmic nucleosomes or by propidium iodide (PI) staining. Cell death in human islets was studied by insulin and cleaved caspase-3 staining. To study single beta cells, islets were mildly dispersed using trypsin. Single islet cells were cytospinned to microscope slides, fixed in 4% paraformaldehyde, blocked, stained using insulin (1/50 dilution) and cleaved caspase-3 (1/50 dilution) antibodies and analysed under a Zeiss Axiovert 40CFL microscope (Carl Zeiss AG, Oberkochen, Germany).

Three micron thick sections from mammary glands of both untreated and DMBA-treated mice were stained with routine hematoxylin and eosin stains (H&E) as described before [31 (link)]. The stained sections were viewed and studied using Zeiss Axiovert 40 CFL microscope, Carl Zeiss LLC (Thornwood, NY) by expert histopathologists. The adequacy of the sample in each case was checked on the semi-thin sections. The thin sections were stained with uranyl acetate and lead citrate then all the sections were examined and photographed by the same histopathologist.

Chemotaxis assay was performed in 24-well Corning^® Costar^® transwell chambers with polycarbonate membranes with pore sizes of 5 μm (Fisher Scientific, Rockville, USA). Equivalent numbers (1 × 10⁵) of gene-modified MSCs were either housed in starPEG-heparin cryogels functionalized with different RGD peptide concentrations (40 μg/ml, 80 μg/ml or 160 μg/ml) or seeded in 2D in the upper chambers in complete RPMI media. The lower compartments were filled either with complete media, or 150 ng/ml SDF-1α (Sigma-Aldrich), or 100 ng/ml TNF-α (PeproTech, Hamburg, Germany) as chemoattractants. Samples were incubated in normoxic incubator for 20 h at 37 °C. Subsequently, the cells from the top side of the membrane insert were removed and migrated cells on the bottom side of the membrane were fixed with 4% formaldehyde (Sigma-Aldrich), stained with 1% crystal violet (Sigma-Aldrich) for 10 min and then visualized using a Zeiss Axiovert 40 CFL microscope equipped with an AxioCam HR camera (both Zeiss).

After deparaffinization and rehydration, sections were soaked in citrate buffer (10 mm citric acid, pH = 6.0, Jiancheng) at 60°C for 12 h to repair the antigen for immunohistochemistry (IHC) and immunofluorescence (IF) staining. Sections were incubated with 3% hydrogen peroxide for 15 min and blocked with 1% sheep serum for 1 h. Then, they were incubated with specific primary antibodies: anti‐COLII (ab188570, Abcam, Cambridge, MA, USA), anti‐ACAN (A11691, Abclonal, Wuhan, China), anti‐MMP13 (A16920), anti‐ADAMTS5 (A2836), anti‐SIRT3 (A20805), and anti‐COX4I2 (ab70112) at 4 °C overnight. For IHC analysis, sections were incubated with horseradish peroxidase (HRP)‐conjugated secondary antibodies, stained with 3,3′‐diaminobenzidine (Vector Laboratories, Burlingame, CA, USA) and counterstained with hematoxylin. For IF analysis, sections were stained with Alexa 488 or Alexa 594 dye‐labeled secondary antibodies (Abcam), and with 4,6‐diamidino‐2‐phenylindole for nuclear counterstaining. Images were captured with a Zeiss Axiovert 40CFL microscope (Zeiss, Oberkochen, Germany). Positively stained cells were quantified using an ImageJ software (National Institutes of Health, Bethesda, MD, USA). The corresponding colocalization ratio based on the Pearson's correlation (Rr) and overlap coefficient (R) between SIRT3 and COX4I2 were calculated by ImageJ analysis.

CD14+ monocytes were seeded on glass cover-slips in 96-well plates and exposed to LCPUFAs (40 μM) or vehicle control in the presence of differentiation factors. PlasDIC images were obtained with a Zeiss Axiocam MRc5 camera attached to a Zeiss Axiovert 40 CFL microscope (Carl Zeiss AG, Oberkochen, Germany).