Autophagy assay cell type - NRK-49F

As autophagy is a multi-step process which includes not just the formation of autophagosomes, but most importantly, flux through the entire system, including the degradation upon fusion with lysosomes, which makes it quite challenging for detection. There are several methods for detection in mammalian cells, including immunoblotting analysis of LC3 and p62 and detection of autophagosome formation/maturation by fluorescence microscopy, Currently, there is no single “gold standard” for determining the autophagic activity that is applicable in every experimental context, hence it is recommended to go for the combined use of multiple methods to accurately assess the autophagic activity in any given biological setting.

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Found 3 matching solutions for this experiment

Protocol tips
Three-μm thickness kidney cryosections were fixed for 15 min in 4% paraformaldehyde, followed by permeabilization with 0.2% Triton X-100 in 1× phosphate-buffered saline (PBS) for 5 min at room temperature. After blocking with 2% donkey serum for 60 min, the slides were immunostained with the primary antibodies. Cells cultured on coverslips were washed twice with cold 1× PBS and fixed with cold methanol/acetone (1:1) for 10 min at −20 °C. After three times of extensive washing with 1× PBS, the cells were treated with 0.1% Triton X-100 for 5 min, blocked with 2% normal donkey serum in 1× PBS buffer for 40 min at room temperature, and incubated with antibodies
Downstream tips
Cells were also stained with 4′,6-diamidino-2-phenylindole to visualize the nuclei. Slides were viewed with a Nikon Eclipse 80i epi-fluorescence microscope equipped with a digital camera or Zeiss LSM710 (Zeiss).
Protocol tips
- Incubate membrane with 5% non-fat milk for 1 h at room temperature before adding primary antibody.

- Dilute Ab at 1:50 and incubate for overnight at 4C.
Beclin-1 Antibody

Cell Signaling Technology

Protocol tips
- Before adding primary antibody, incubate membrane with 5% non-fat milk for 1 h at room temperature.

- Incubate primary antibody overnight at 4°C
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