Autophagy assay cell type - BRL-3A

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 2 matching solutions for this experiment

Anti-LC3A/B antibody (ab62721)

Abcam

Upstream tips	Protocol tips	Downstream tips
	Rat liver tissue was fixed with 4% formaldehyde followed by routine dehydration and paraffin embedding. Paraffinembedded tissue was sliced into 4 um sections, followed by dewaxing with xylene and gradient alcohol hydration in strict accordance with the kit instructions (PV-900, Beijing Zhongyu Jinqiao Biotechnology Co., Ltd.). Beclin1 and LC3 antibodies were purchased from Abcam Corporation, UK, and used at 1:1000 dilution. For negative controls, the primary antibodies were replaced with PBS. The results showed that Beclin1 and LC3 were present in both the cytoplasm and nucleus, the positive expression were light yellow to yellowish brown granules , and the areas with brownish yellow color were positive. Five fields were randomly selected for each section (magnification 400X), 100 cells were counted and the percentage of positive cells was calculated to indirectly estimate the expression levels.

Protocol tips
Rat liver tissue was fixed with 4% formaldehyde followed by routine dehydration and paraffin embedding. Paraffinembedded tissue was sliced into 4 um sections, followed by dewaxing with xylene and gradient alcohol hydration in strict accordance with the kit instructions (PV-900, Beijing Zhongyu Jinqiao Biotechnology Co., Ltd.). Beclin1 and LC3 antibodies were purchased from Abcam Corporation, UK, and used at 1:1000 dilution. For negative controls, the primary antibodies were replaced with PBS. The results showed that Beclin1 and LC3 were present in both the cytoplasm and nucleus, the positive expression were light yellow to yellowish brown granules , and the areas with brownish yellow color were positive. Five fields were randomly selected for each section (magnification 400X), 100 cells were counted and the percentage of positive cells was calculated to indirectly estimate the expression levels.

Protocol tips

Rat liver tissue was fixed with 4% formaldehyde followed by routine dehydration and paraffin embedding. Paraffinembedded tissue was sliced into 4 um sections, followed by dewaxing with xylene and gradient alcohol hydration in strict accordance with the kit instructions (PV-900, Beijing Zhongyu Jinqiao Biotechnology Co., Ltd.). Beclin1 and LC3 antibodies were purchased from Abcam Corporation, UK, and used at 1:1000 dilution. For negative controls, the primary antibodies were replaced with PBS. The results showed that Beclin1 and LC3 were present in both the cytoplasm and nucleus, the positive expression were light yellow to yellowish brown granules , and the areas with brownish yellow color were positive. Five fields were randomly selected for each section (magnification 400X), 100 cells were counted and the percentage of positive cells was calculated to indirectly estimate the expression levels.

Manufacturer protocol Publication protocol 1 Relevant paper

Beclin-1 Antibody

Cell Signaling Technology

Upstream tips	Protocol tips	Downstream tips
- Do not aliquot the antibody	- Dilute Primary Ab at 1:1000

Upstream tips
- Do not aliquot the antibody
Protocol tips
- Dilute Primary Ab at 1:1000

Manufacturer protocol Publication protocol 1 Relevant paper

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