Protocol - Autophagosomes staining with Anti-LC3B-II antibodies in epithelial cells
LC3B-II is a protein stably associate with the autophagosome membranes. It is considered a useful and sensitive marker for distinguishing autophagy in mammalian tissue and cultured cells.
Category:
In situ hybridization and Immunochemistry
Creation date: Jul 19, 2013
Last revision: Mar 02, 2016
Author(s):
G Calì, A Mascia
Contact
Name: Anna Mascia
Address: Institute of Experimental Endocrinology and Oncology “G. Salvatore”, CNR via Pansini 5, 80131 Naples Italy
Phone: +3908107464575 / 3237
Email: a.mascia@ieos.cnr.it
Figure legend:
Immunofluorescence staining with rabbit polyclonal anti-LC3B-II antibodies revealed by Alexa Fluor 488 goat anti-rabbit secondary antibody in FRT cell line. DRAQ5 nuclear staining (Mascia et al., 2015).
Steps
| Description | Temperature | Time | Note |
|---|---|---|---|
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Place one 12 mm diameter glass coverslip per well in 24-well plate. Seed cells on coverslip with complete medium
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37°C, 5% CO2 in a humidified incubator
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48 hrs before experiment
|
60% confluent cells
|
|
Fix with Methanol
|
-20°C
|
10 min
|
Methanol prestored -20°C
|
|
Permeabilize with Acetone
|
-20°C
|
1 min
|
Acetone prestored -20°C
|
|
Wash with TBS 1X
|
RT
|
3 x 5 min
|
Tris-Buffered Saline ( 20mM Tris-HCl, pH7,5; 150mM NaCl)
|
|
Block with blocking solution
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RT
|
60 min
|
1% BSA in TBS 1X
|
|
Incubate the coverslip with 20 μl primary antibody :
|
RT in humidified chamber containing a rectangle of parafilm
|
60 min
|
Put Ab onto parafilm. Turn upside down the coverslip. Keep the cells to direct contact with Ab
|
|
LC3B-II antibody diluted 1:400 in blocking solution
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Polyclonal antibody from Cell Signaling Technology
|
||
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Wash with TBS 1X
|
RT
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3 x 5 min
|
coverslip with cells side up
|
|
Incubate with 20 μl secondary antibody :
|
RT in humidified chamber containing a rectangle of parafilm
|
30 min
|
Put Ab onto parafilm. Turn upside down the coverslip. Keep the cells to direct contact with Ab
|
|
Alexa Fluor diluted 1:200 in blocking solution
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Alexa Fluor goat anti-rabbit
|
||
|
Wash with TBS 1X
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RT
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5 x 5 min
|
coverslip with cells side up
|
|
Incubate with 20 μl the DNA intercalator :
|
RT in humidified chamber containing a rectangle of parafilm
|
10 min
|
Put DRAQ5 onto parafilm.Turn upside down the coverslip. Keep the cells to direct contact with DRAQ5
|
|
DRAQ5 diluted 1:3000
|
Alexis Corp., Lausen, Switzerland
|
||
|
Wash with TBS 1X
|
RT
|
3 min
|
coverslip with cells side up
|
|
Place 2 μl mounting medium on microscope slide
|
RT
|
1:1 PBS Glycerol
|
|
|
Turn upside down the coverslip. Keep the cells to direct contact with mounting medium
|
|||
|
Collect image or store at 4°C in dark box
|
Quality validation:
Yes
Validation info
The protocol has been published in a peer-reviewed journal.
Related ModelSystems:
Solutions
-
TBS 1X ( 100ml )
Reagents F-concentration Quantity I-concentration Tris-HCl pH7,520mM10ml200mMNaCl150mM10ml1,5MH2Odd up to 100ml
