For confocal laser-scanning microscopy, the organoids or the tissue was fixed and embedded in agarose or in OCT compound 4583 (Sakura) as already described (37 (link)). Sections of 150 μm (agarose) or 10 μm (OCT) were obtained by using an HM650V vibratome (Thermo Fisher) or a CM3050S cryostat (Leica), respectively. For antigen retrieval, slides were steamed for 20 min in 10 mM citrate buffer (pH 6) and cooled for 30 min. Rabbit anti-cleaved caspase-3 (1:100; Cell Signaling Technology; Asp175), chicken anti-GFP (1:500; Abcam; ab13970), rabbit anti-LC3B (1:200; Abgent; AP1802a), rabbit anti-p65 (1:100; Abcam; 7970), mouse anti-Olfm4 (1:200; Cell Signaling Technology; D6Y5A), phalloidin 568 (Thermo Fisher), and corresponding secondary antibodies (Alexa Fluor 488, 568, or 647; Thermo Fisher) were used. DNA was stained by DAPI (1 μg/mL; Thermo Fisher). The sections were mounted with ProLong Gold Antifade Reagent (Thermo Fisher). Images were acquired using an IX-81 (Olympus) or Opterra swept-field confocal microscope (Bruker) equipped with thermic chamber and CO2 control. Postacquisition image analysis was performed with Fiji 2.0.0 software.
Ap1802a
Manufactured by Abcam
AP1802a is a lab equipment product. It is a device used for a specific laboratory function. No further details are available at this time.
Automatically generated - may contain errors
Lab products found in correlation
Alexa fluor 488, by Thermo Fisher Scientific (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (2 mentions)
Hm650v vibratome, by Thermo Fisher Scientific (1 mentions)
Opterra swept field confocal microscope, by Bruker (1 mentions)
Phalloidin 568, by Thermo Fisher Scientific (1 mentions)
Asp175, by Abcam (1 mentions)
Prolong gold antifade reagent, by Thermo Fisher Scientific (1 mentions)
Cm3050s cryostat, by Leica (1 mentions)
Draq5, by Cell Signaling Technology (1 mentions)
D1306, by Thermo Fisher Scientific (1 mentions)
2 protocols using ap1802a
1
Confocal Microscopy of Organoids and Tissues
2
Quantifying Lipid Droplets in Cryosections
Check if the same lab product or an alternative is used in the 5 most similar protocols
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Part of the median lobe was embedded in tissue-Tek (VWR, 25608-930) and kept at −80 °C till cryosectioning. Cryosections (5μm thickness) were fixed in 4% PFA for 15 min, then briefly washed with running tap water and 60% isopropanol. Then cryosections were incubated with Bodipy (Life Technologies, D-3922, 1:250), LC3 (Abgent, AP1802a; 1:100), LAMP1 (Abcam, ab24170, 1:100), or PLIN3 (ProSci, 3883, 1:100) for 30 min. After rinsing steps with 60% isopropanol, cryosections were incubated for 30 min with donkey anti-rabbit cy3 (Diannova, 711-166-152, 1:200). Finally, the slides were incubated either with Draq5 (Cell Signaling Technology, 4084 L, 1:5000) or DAPI (Invitrogen, D1306, 1:1000). Two tile scans of 9 images each per mouse for quantification were acquired using confocal microscopy (Leica SP8, UMM-Core facility Mannheim, Germany). Lipid droplets quantification was performed using ImageJ (https://imagej.nih.gov/ij/ ) on tile scans.
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