CYTO-ID® Autophagy detection kit

Protocol tips

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Islets cells pretreated with rapamycin (500 nmol/L) can be used as positive control	After staining wash the cells with 1X assay buffer and transfer it to the glass slide for imaging. Add dye and incubate for 30 min at 37°C

Upstream tips
Islets cells pretreated with rapamycin (500 nmol/L) can be used as positive control
Protocol tips
After staining wash the cells with 1X assay buffer and transfer it to the glass slide for imaging. Add dye and incubate for 30 min at 37°C

Publication protocol

Pancreata were dissected and fixed in 10% PBS-formalin for 24 h. After paraffin embedding and sectioning (5 μm), tissues were stained with hematoxylin-eosin (H-E). For immunofluorescence (IF) and immunohistochemistry (IHC), paraffin-embedded sections were incubated with primary antibodies for 24 h at 4°C and subsequently decorated with secondary antibodies for 1 h at room temperature (RT). Antibodies used were a polyclonal anti-mitoNEET antibody (1:250; Santa Cruz Biotechnology, Dallas, TX), a guinea pig anti-swine insulin antibody (1:500; Dako, Carpinteria, CA), a rabbit antiglucagon antibody (1:250; Zymed, Grand Island, NY), and a polyclonal goat beclin-1 antibody (1:250; Santa Cruz Biotechnology). For IHC, sections were incubated with biotinylated secondary antibodies (anti-guinea pig antibody [1:500], anti-rabbit antibody [1:200], or anti-goat antibody [1:500]; Dako) for 1 h at RT, then reactions were developed using DAB (3,3'-diaminobenzidine) chromogen and DAB substrate buffer (Dako). All images were acquired with an Olympus FSZ100 light microscope or a Zeiss AxioObserver epifluorescence microscope. For microtubule-associated protein light chain 3 (LC3-II) IF staining, isolated islets were treated for 30 min at 37°C with a mixture containing CYTO-ID Green Detection Reagent Hoechst 33342 Nuclear Stain per manufacturer protocol (CYTO-ID Autophagy Detection Kit; Enzo Life Sciences, Inc., Farmingdale, NY). As a positive control, WT islets were pretreated with rapamycin (500 nmol/L). Islets were then washed twice with 1× assay buffer and transferred to a glass microscope slide. Autophagic signal was imaged by using confocal microscopy in the fluorescein isothiocyanate 488-nm excitable green fluorescent detection range (Leica TCS SP5 confocal microscope).

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Manufacturer protocol

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