Transduced cells were then incubated cisplatin dna in regular M medium, and cells were cultured until d 7. For transduction of LLC-PK1 cells, cells were cultured with retrovirus containing medium with polybrene 8 ��g/ml, for 24 h. One day later, cells were selected with puromycin 3 ��g/ml for 72 h. To assess autophagy, cells were incubated with bafilomycin A1 with rapamycin (200 nM, 4 h) or bafilomycin A1 with chloroquine (50 ��M, 4 h). Alternatively cells were amino acid starved in HEPES-buffered HBSS for 1, 2, or 4 h and then fixed with 2% PFA for 5 min. Immunofluorescence detection and microscopy Mouse and rat tissues were prepared either by perfusion fixation using 4% paraformaldehyde, l-lysine, or periodate (PLP) or by perfusion of organs with ice-cold PBS, followed by dissection of organs and fixing in PLP for 2 h, followed by 18% sucrose for 16 h prior to cryosectioning at 5 ��m as described previously (18).
For immunofluorescence, rat anti-mouse CD68 (1:200), mouse anti-rat CD68 antibody (ED1; 1:200; ABD-Serotec, Kidlington, UK), rat anti-F4/80 (1;200; eBioscience, San Diego, CA, USA), mouse anti-vimentin antibodies (1:200; Sigma), anti-human LAMP-1 hybridoma supernatant (1:200; Developmental Studies Hybridoma Bank, University of Iowa, Iowa City, IA, USA), anti-human EEA-1 antibodies (1:200; Santa Cruz Biotechnology, Santa Cruz, CA, USA), rabbit anti-ATG12 (1:100), rabbit anti-LC3 (1:200; (Abgent, San Diego, CA, USA), anti-Gpnmb cyto (1:500), or anti-Gpnmb ecto (1:500) were applied to cryosections in blocking medium for 2 h at room temperature, followed by washing.
Cy2- or Cy3-conjugated affinity-purified secondary antibodies were applied in a blocking medium (Jackson ImmunoResearch, West Grove, PA, USA) using methods described previously (18). After washing, slides were mounted Carfilzomib with Vectashield containing DAPI (200 nM). LysoTracker Red (Molecular Probes) was applied to live epithelial cells or Ms in culture for 0.5�C2 h at 37��C at a concentration of 50�C200 nM. Filipin (Sigma) 50 ng/ml was applied to fixed cells for 2 h and 25��C using methods adapted from established methods (24), and images were visualized using fluorescence microscopy. Confocal images were generated by taking 30 or 40 3-channel fluorescent image stacks (Nikon TE2000 microscope; Nikon, Tokyo, Japan) 0.2 ��m apart, then generating confocal images using deconvolution software (Autodeblur; Media Cybernetics, Bethesda, MD, USA) as described previously (18). For Gpnmb detection in kidney sections, confocal images were generated using a Nikon C1 D-Eclipse confocal microscope. Projection images were generated from 10 Z-stack images that were acquired at 0.1-��m steps. To allow comparison between sections, all confocal settings were kept constant between sections.
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