Spleens excised from mice 2 or 3 weeks after MAC infection were embedded in cryomolds, frozen in liquid nitrogen, cut as serial sections in 14- to 16-μm thickness, and fixed with paraformaldehyde followed by serial treatments with acetic acid/ethanol (1:2) and 1% BSA/0.05% Tween 20 in phosphate-buffered saline (PBS). The resultant sections were reacted with rabbit anti-mouse NOS2 Ab (class IgG: Santa Cruz Biotechnology) or rabbit anti-mouse Arg-1 Ab (IgG: Santa Cruz Biotechnology), followed by staining with Alexa Fluor 488- conjugated donkey anti-rabbit IgG Ab (Molecular Probes). Fluorescence microscopy was performed using an FV1000D IX81 confocal microscope (Olympus) and Eclipse 80i fluorescence microscope (Nikon). The remaining sections were subjected to HE and Ziehl-Neelsen staining.
Fv1000 d ix81 confocal microscope
Manufactured by Olympus
Sourced in Japan
The FV1000-D IX81 is a confocal microscope designed for high-resolution imaging of biological samples. It features a motorized research-grade inverted microscope and a confocal laser scanning unit for capturing detailed fluorescence images.
Automatically generated - may contain errors
Lab products found in correlation
Eclipse 80i fluorescence microscope, by Nikon (1 mentions)
Prolong gold antifade reagent with dapi, by Thermo Fisher Scientific (1 mentions)
Cytospin 4, by Thermo Fisher Scientific (1 mentions)
Anti lc3, by Cell Signaling Technology (1 mentions)
Cytoslides, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 488 goat anti mouse igg, by Thermo Fisher Scientific (1 mentions)
Anti flag m2, by Merck Group (1 mentions)
Alexa fluor 594 anti rabbit igg antibodies, by Thermo Fisher Scientific (1 mentions)
Permafluor mountant, by Thermo Fisher Scientific (1 mentions)
Blocking reagent, by Roche (1 mentions)
Pacgfp1 c1, by Takara Bio (1 mentions)
3 protocols using fv1000 d ix81 confocal microscope
1
Immunohistochemistry Analysis of Infected Mouse Spleens
2
Immunofluorescence Assay for Autophagy
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Cells were treated with or without 300 nM 17-AAG and/or 100 nM BAFA1 for 6 h, and 1 × 106 cells were collected into 1.5 mL microcentrifuge tubes by centrifugation at 200 × g for 5 min at 4° C. After washing cells with ice-cold PBS, they were fixed with 3.7% formalin/PBS for 15 min at room temperature, permeabilized with 0.2% Triton X-100/PBS for 15 min at room temperature, transferred to FBS-coated 0.2-mL PCR tubes, and then blocked in 3% BSA/PBS for 30 min at room temperature. Cells were incubated with anti-FLAG M2 (50 μg/mL, Sigma-Aldrich) and anti-LC3 (1:50 dilution, Cell Signaling Technology) antibodies overnight at 4° C, followed by incubation with secondary antibodies, Alexa Fluor® 488 goat anti-mouse IgG and Alexa Fluor® 594 anti-rabbit IgG antibodies (Invitrogen), for 2 h at room temperature. Cells were washed three times with PBS, plated on cytoslides (Thermo Fisher Scientific Inc., Waltham, MA) using Cytospin®4 (Thermo Fisher Scientific Inc.), and mounted with Prolong® Gold antifade reagent with DAPI (Invitrogen). Acquisition of images was performed using a FV1000-D IX81 confocal microscope (Olympus Corp., Tokyo, Japan). Confocal 2-D TIFF images were merged using Adobe® Photoshop CC software (Adobe Systems Inc., San Jose, CA).
3
PPARα Localization in COS-7 Cells
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The COS-7 cells were cultured on the 8-well chamber plates at a cell density of 2.5 × 104 cells/well for 24 h. The COS-7 cells were co-transfected with 0.2 µg of pAcGFP1-C1 (Clontech) and 0.4 µg of pcDNA3-PPARα-WT or variant expression constructs with 50 µM WY14643 for 24 h. The cells were fixed with 4% paraformaldehyde in 0.01 M phosphate-buffered saline (PBS) for 15 min and washed with PBS. The fixed cells were permeabilized with 0.2% Triton X-100 and were blocked with 1% Blocking Reagent (Roche Diagnostics, Mannheim, Germany). The cells were then incubated with the primary antibody against PPARα (Santa Cruz Biotechnology, Santa Cruz, CA, USA) for 1 h at room temperature. The cells were washed with PBS and were incubated with the Alexa Fluor 594-conjugated secondary antibody. The cells were then washed with PBS and incubated with 4ʹ,6-diamidino-2-phenylindole (DAPI, Roche) for nucleus staining. Next, the cells mounted with PermaFluor Mountant (Thermo Fisher Scientific, Waltham, MA, USA), and the images were captured under an FV1000-D IX81 confocal microscope (Olympus, Tokyo, Japan). The images were analyzed using the ImageJ software (https://imagej.nih.gov/ij/ ).
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