In order to verify exosomal uptake by recipient cells and the intracellular localization, confocal images were recorded. For this, 15x103 SKOV3 cells were seeded into Nunc™ Lab-Tek™ II Chamber Slides™ (Nunc, Roskilde, Denmark) and cultured with exosomes overexpressing CD63-GFP. After 24 h, SKOV3 cells were fixed with 4% PFA and permeabilised using 0.1% TritonX. Afterwards cells were blocked with a 3% BSA, 0.1% Tween20 and 0.3 M Glycin solution. Organelles were stained with the specific antibodies Anti-EEA1 (Abcam #ab2900; 1:200), Anti-Lamp1 (Abcam #ab24170; 1:200), Anti-GM130 (BD BioScience, Heidelberg, Germany, #610822; 1:200) and Anti-GRP78 (Abcam #21685; 1:300). As secondary antibodies Anti-Rabbit AlexaFluor594 (Jackon ImmunoResearch, Cambridge, UK, #711-585-152; 1:500) and Anti-mouse Rhodamine Red-X (Jackson ImmunoResearch #115-295-206; 1:100) were used. Isotype controls were purified mouse IgG1 ĸ isotype control (BD BioScience #554121; 1:200) and rabbit IgG, polyclonal isotype control (Abcam #ab37415; 1:40). Cells were mounted with Prolong® Diamond Antifade Mountant medium (Invitrogen, Darmstadt, Germany) and covered with a glass slide. Images were recorded with the confocal microscope Zeiss LSM 710 (Carl Zeiss, Oberkochen, Germany).
Prolong diamond antifade mountant medium
Manufactured by Thermo Fisher Scientific
Sourced in Germany
Prolong® Diamond Antifade Mountant medium is a ready-to-use, aqueous-based mounting medium designed for fluorescence microscopy. It is formulated to maintain the brightness and photostability of fluorescent labels.
Automatically generated - may contain errors
Lab products found in correlation
Lsm 710, by Zeiss (1 mentions)
Nunc lab tek 2 chamber slide, by Thermo Fisher Scientific (1 mentions)
Anti gm130, by BD (1 mentions)
Ab2900, by Abcam (1 mentions)
Ab37415, by Abcam (1 mentions)
Anti mouse rhodamine red x, by Jackson ImmunoResearch (1 mentions)
Anti grp78, by Abcam (1 mentions)
Lsm 800, by Zeiss (1 mentions)
Goat serum, by Thermo Fisher Scientific (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Biotium (1 mentions)
Airyscan fluorescent confocal microscope, by Zeiss (1 mentions)
Tcs sp5, by Leica (1 mentions)
Mitotracker, by Thermo Fisher Scientific (1 mentions)
Ab186734, by Abcam (1 mentions)
Sp5 confocal microscope, by Leica (1 mentions)
Ab150073, by Abcam (1 mentions)
4 protocols using prolong diamond antifade mountant medium
1
Visualizing Exosomal Uptake and Intracellular Localization
2
Immunofluorescence Staining of Cryosectioned Brains
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Brains were cryosectioned at 12 μm per slice and mounted on a charged slide. Following thawing in a humidified chamber, tissues were incubated in blocking solution consisting of 5% goat serum (Gibco, 16210) and 0.2% Triton X-100 for one hour at room temperature. Sections were then incubated with primary antibody diluted in blocking solution overnight at 4°C in a humidified chamber. Slides were then washed three times with PBS for 15 minutes followed by incubation in secondary antibody diluted in blocking solution for one hour at room temperature. Slides were washed three times to remove secondary antibody and were then stained with 4’,6-diamindino-2-phenylindole (DAPI; Biotium, 40011) diluted in PBS for 20 minutes at room temperature, followed by another wash. Sections were cover-slipped with Prolong Diamond Antifade Mountant medium (Invitrogen, P36930). Slides were allowed to dry and images were acquired using Airyscan fluorescent confocal microscope (Carl Zeiss, LSM 800).
3
Ir Complex Uptake and Mitochondrial Localization
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MDA-MB-231 cells were seeded
on 35 mm glass-bottom confocal culture dishes (Mattek Co., MA, USA)
at 1 × 105 cells/dish density and incubated overnight.
Then, the cells were treated with tested compounds (5 μM) and
incubated for 5 h. After incubation, cells were co-stained with MitoTracker
(Thermo Fisher Scientific, Waltham, MA, USA). Samples were fixed with
3.7% formaldehyde, mounted with ProLong Diamond Antifade Mountant
medium (Thermo Fisher Scientific), and then analyzed on a confocal
laser-scanning microscope Leica TCS SP5 (Leica Microsystems GmbH,
Wetzlar, Germany). The investigated Ir complexes were excited at 355
nm. Samples were scanned sequentially. Colocalization analysis of
acquired images was performed using ImageJ software. Briefly, the
Pearson coefficient of correlation (PCC) was measured for entire images
by default, and the Costes regression method was used to estimate
the threshold. Values of PCC are expressed as the mean from two independent
experiments ± SDs.
on 35 mm glass-bottom confocal culture dishes (Mattek Co., MA, USA)
at 1 × 105 cells/dish density and incubated overnight.
Then, the cells were treated with tested compounds (5 μM) and
incubated for 5 h. After incubation, cells were co-stained with MitoTracker
(Thermo Fisher Scientific, Waltham, MA, USA). Samples were fixed with
3.7% formaldehyde, mounted with ProLong Diamond Antifade Mountant
medium (Thermo Fisher Scientific), and then analyzed on a confocal
laser-scanning microscope Leica TCS SP5 (Leica Microsystems GmbH,
Wetzlar, Germany). The investigated Ir complexes were excited at 355
nm. Samples were scanned sequentially. Colocalization analysis of
acquired images was performed using ImageJ software. Briefly, the
Pearson coefficient of correlation (PCC) was measured for entire images
by default, and the Costes regression method was used to estimate
the threshold. Values of PCC are expressed as the mean from two independent
experiments ± SDs.
4
Immunohistochemistry analysis of TOMM20 in skeletal muscle and liver
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Immunohistochemistry assay was performed on deparaffinized Wistar or WAR soleus skeletal muscle and liver tissues (paraffin sections 10 m thick). To expose target proteins, heat-induced antigen retrieval was performed using 10 mM sodium citrate (pH 6.0) buffer microwaved for 8 -15 min. After antigen retrieval, tissues were permeabilized with 0.3% (vol/vol) Triton X-100 and blocked with 100 mM glycine, and then 3% (wt/vol) BSA-PBS at room temperature. Tissues were then probed at a concentration of 2 g/ml with a rabbit monoclonal antibody recognizing TOMM20 (EPR15581), a mitochondrial marker (Abcam ab186734) or without primary antibody (negative control) overnight at 4°C in a humidified chamber. Tissues were washed extensively with PBS and probed with 2 g/ml secondary antibody Alexa fluor donkey anti-rabbit 594 (Abcam ab150073) IgG. The slides were mounted on ProLong Diamond Antifade Mountant medium with DAPI (Thermo Fisher Scientific, P36962), and the images were collected on a Leica SP5 confocal microscope, with a ϫ63 objective. The Icy BioImage open source software from the Pasteur Institute (http://icy.bioimageanalysis.org/) was used for image processing.
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