RAW 264.7 macrophages (0.2 x 106 cells) were seeded on glass coverslips in a 24 well plate and left for adhesion overnight. Cells were then incubated with nanoemulsion (30 μL) in culture media (1 mL) for two hours at 37 °C. Cell were then washed with DPBS and fixed with 4% paraformaldehyde (PFA) for 20 min at room temperature (RT) and stained with DAPI for nuclei visualization. For kinetic studies, cells were incubated with nanoemulsion (30 μL in 1 mL medium) for 5, 15, 30 and 60 min at 4 and 37 °C, fixed with 4% PFA, and stained with DAPI. After washing with DPBS, coverslips were mounted using Diamond anti-fading medium (Invitrogen, Grand Island, NY). Fluorescence was monitored using a Zeiss Apotome system equipped with a Zeiss HPO PL APO 63x oil immersion lens (numerical aperture 1.4-0.6). For colocalization with acidic compartments, cells were seeded on a glass bottom dishes and left for adhesion. Cells were then incubated with nanoemulsion (30 μL in 1 mL medium) for 45 min at 37 °C, and Lysotracker Green DND-26 (1 μM) (Invitrogen, Grand Island, NY) was added for 15 min at 37 °C. Cells were washed with DPBS and immediately imaged using a Zeiss Apotome system equipped with a Zeiss HPO PL APO 63x oil immersion lens (numerical aperture 1.4-0.6).
Diamond anti fading medium
Manufactured by Thermo Fisher Scientific
Diamond anti-fading medium is a mounting medium designed to protect fluorescently-labeled samples from photobleaching. It is a solvent-based solution that helps maintain the brightness and stability of fluorescent signals during microscopy imaging and analysis.
Automatically generated - may contain errors
Lab products found in correlation
Lysotracker green dnd 26, by Thermo Fisher Scientific (1 mentions)
Apotome system, by Zeiss (1 mentions)
Hpo pl apo 63x oil immersion lens, by Zeiss (1 mentions)
Microtome, by Leica camera (1 mentions)
Nuance software, by PerkinElmer (1 mentions)
Anti goat alexa 594, by Thermo Fisher Scientific (1 mentions)
Rat anti mouse cd68, by Bio-Rad (1 mentions)
Anti rat alexa 488, by Thermo Fisher Scientific (1 mentions)
2 protocols using diamond anti fading medium
1
Macrophage Uptake Kinetics of Nanoemulsions
2
Histological and Immunofluorescent Analysis of CFA-Induced Inflammation
Check if the same lab product or an alternative is used in the 5 most similar protocols
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Male and female mice were sacrificed after 40 days post CFA injection, and both of the right and left paws were harvested. Tissues were prepared for histology with hematoxylin and eosin (H&E) and immunostaining. The excised tissue and organ samples were fixed in 4% PFA solution, embedded into paraffin, and cut into 10 μm sections using microtome (Leica, Buffalo Grove, IL). The sections were stained with H&E according to standard protocols. For immunostaining; non-specific binding was blocked with Dako serum free protein for 15 min at room temperature. Rat anti-mouse CD68 (Bio-Rad, Hercules, CA) (1/200 dilution in DPBS/0.1% BSA/0.05% Tween-20) and goat anti-mouse COX-2 antibodies (Novus, Centennial, CO) (at 1/50 dilution in PBS/0.1% BSA/0.05% Tween-20) were added overnight at 4 °C followed by a secondary antibody, anti-rat-Alexa 488 and anti-goat- Alexa 594 (Invitrogen, Grand Island, NY) (1/300 dilution in PBS/0.1% BSA/0.05% Tween-20) for 1 h at RT. After washing in DPBS/0.05% Tween-20, coverslips were mounted using Diamond anti-fading medium (Invitrogen, Grand Island, NY). Immunofluorescence staining was monitored using an Olympus BX63 fluorescence microscope with a multispectral camera by CRI (Perkin Elmer) and Nuance software.
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