Imaging Autophagy

Eleftherios Karanasios1, Eloise Stapleton2, Maria Manifava1, Nicholas T. Ktistakis1

1 Signalling Programme, The Babraham Institute, Cambridge, 2 MRC Group, Cardiff School of Biosciences, Cardiff University, Cardiff
Publication Name:  Current Protocols in Cytometry
Unit Number:  Unit 12.34
DOI:  10.1002/0471142956.cy1234s69
Online Posting Date:  July, 2014
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Abstract

Autophagy is a membrane‐trafficking pathway activated to deliver cytosolic material for degradation to lysosomes through a novel membrane compartment, the autophagosome. Fluorescence microscopy is the most common method used to visualize proteins inside cells, and it is widely used in the autophagy field. To distinguish it from the cellular background, the protein of interest (POI) is either fused with a genetically encoded fluorescent protein or stained with an antibody that is conjugated to an inorganic fluorescent compound. Genetic tagging of the POI allows its visualization in live cells, while immunostaining of the POI requires the fixation of cells and the permeabilization of cell membranes. Here we describe detailed protocols on how to visualize autophagy dynamics using fluorescence microscopy in live and fixed cells. We discuss the critical parameters of each technique, their advantages, and why the robustness is increased when they are used in tandem. Curr. Protoc. Cytom. 69:12.34.1‐12.34.16. © 2014 by John Wiley & Sons, Inc.

Keywords: autophagy; omegasomes; ULK1; fluorescence microscopy; live‐cell imaging

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Immunofluorescence Microscopy of Autophagy with Formaldehyde Fixation
  • Alternate Protocol 1: Immunofluorescence Microscopy of Autophagy with Methanol Fixation
  • Basic Protocol 2: Time‐Lapse Fluorescence Microscopy of Autophagosome Formation
  • Reagents and Solutions
  • Commentary
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Immunofluorescence Microscopy of Autophagy with Formaldehyde Fixation

  Materials
  • 100% ethanol
  • HEK‐293 cells (ATCC no. CRL‐1573) at low passage number
  • Complete medium (see recipe)
  • Amino acid starvation medium (see recipe)
  • PP242 (optional; Sigma, cat. no. P0037)
  • Formaldehyde solution (see recipe)
  • Blocking buffer (see recipe)
  • NETgel (see recipe)
  • Permeabilization buffer (see recipe)
  • Primary antibodies (Table 12.34.1)
  • Secondary antibodies (Table 12.34.2)
  • Aqua Poly Mount mounting medium (Polysciences, cat. no. 18606)
  • Phosphate‐buffered saline (PBS; see recipe)
  • 16‐mm round coverslips (VWR, cat. no. 631‐0152)
  • Forceps
  • 12‐well plate
  • Rocker
  • Forceps
  • Glass microscope slides
  • Microscope (wide‐field or confocal)
Table 2.4.1   MaterialsPrimary Antibodies Against Autophagy Proteins that are Used Routinely in our LabSecondary Antibodies Used Routinely in Our Lab Against the Primary Antibodies in Table 12.34.1

Antigen Vendor Cat. no. Fixative Source Dilution
ULK1 Santa Cruz sc‐33182 Formaldehyde Rabbit 1:100
RB1CC1 (FIP200) Proteintech 17250‐1‐AP Formaldehyde Rabbit 1:70
Atg13 Sigma SAB4200100 Formaldehyde Rabbit 1:100
Atg13 Millipore MABC46 Formaldehyde Mouse 1:100
Atg101 Sigma SAB4200175 Formaldehyde Rabbit 1:100
Atg16 MBL PM040 Formaldehyde Rabbit 1:100
WIPI2 AbD Serotec MCA5780GA Formaldehyde Mouse 1:200
LC3B Sigma L7543 Methanol Rabbit 1:200
SQSTM1 (p62) Abnova H00008878‐M01 Formaldehyde Mouse 1:100
Antigen Vendor Cat.no. Comments Dilution
Goat anti‐mouse FITC Jackson ImmunoResearch 115‐095‐062 Fluorescein conjugated; purified IgG 1:100
Goat anti‐mouse TRITC Jackson ImmunoResearch 115‐025‐062 Rhodamine conjugated; purified IgG 1:100
Goat anti‐rabbit FITC Jackson ImmunoResearch 111‐085‐045 Fluorescein conjugated; purified IgG 1:100
Goat anti‐rabbit TRITC Jackson ImmunoResearch 111‐025‐144 Rhodamine conjugated; purified IgG 1:100
Donkey anti‐goat TRITC Jackson ImmunoResearch 705‐025‐147 Rhodamine conjugated; purified IgG 1:100
Donkey anti‐rabbit FITC Jackson ImmunoResearch 711‐095‐152 Fluorescein conjugated; purified IgG 1:100
Goat anti‐mouse Cy2 Jackson ImmunoResearch 115‐225‐146 Cy2 conjugated; purified IgG 1:500
Goat anti‐rabbit Cy2 Jackson ImmunoResearch 111‐225‐144 Cy2 conjugated; purified IgG 1:500
Goat anti‐mouse Alexa Fluor 647 Molecular Probes A21236 Alexa Fluor 647 conjugated; purified IgG 1:500‐1:1000

Table 2.4.2   MaterialsPrimary Antibodies Against Autophagy Proteins that are Used Routinely in our LabSecondary Antibodies Used Routinely in Our Lab Against the Primary Antibodies in Table 12.34.1

Antigen Vendor Cat. no. Fixative Source Dilution
ULK1 Santa Cruz sc‐33182 Formaldehyde Rabbit 1:100
RB1CC1 (FIP200) Proteintech 17250‐1‐AP Formaldehyde Rabbit 1:70
Atg13 Sigma SAB4200100 Formaldehyde Rabbit 1:100
Atg13 Millipore MABC46 Formaldehyde Mouse 1:100
Atg101 Sigma SAB4200175 Formaldehyde Rabbit 1:100
Atg16 MBL PM040 Formaldehyde Rabbit 1:100
WIPI2 AbD Serotec MCA5780GA Formaldehyde Mouse 1:200
LC3B Sigma L7543 Methanol Rabbit 1:200
SQSTM1 (p62) Abnova H00008878‐M01 Formaldehyde Mouse 1:100
Antigen Vendor Cat.no. Comments Dilution
Goat anti‐mouse FITC Jackson ImmunoResearch 115‐095‐062 Fluorescein conjugated; purified IgG 1:100
Goat anti‐mouse TRITC Jackson ImmunoResearch 115‐025‐062 Rhodamine conjugated; purified IgG 1:100
Goat anti‐rabbit FITC Jackson ImmunoResearch 111‐085‐045 Fluorescein conjugated; purified IgG 1:100
Goat anti‐rabbit TRITC Jackson ImmunoResearch 111‐025‐144 Rhodamine conjugated; purified IgG 1:100
Donkey anti‐goat TRITC Jackson ImmunoResearch 705‐025‐147 Rhodamine conjugated; purified IgG 1:100
Donkey anti‐rabbit FITC Jackson ImmunoResearch 711‐095‐152 Fluorescein conjugated; purified IgG 1:100
Goat anti‐mouse Cy2 Jackson ImmunoResearch 115‐225‐146 Cy2 conjugated; purified IgG 1:500
Goat anti‐rabbit Cy2 Jackson ImmunoResearch 111‐225‐144 Cy2 conjugated; purified IgG 1:500
Goat anti‐mouse Alexa Fluor 647 Molecular Probes A21236 Alexa Fluor 647 conjugated; purified IgG 1:500‐1:1000

 aSecondary antibodies are purchased as freeze‐dried powder and reconstituted according to the product specifications sheet.
 bThis list is by no means exhaustive.

Alternate Protocol 1: Immunofluorescence Microscopy of Autophagy with Methanol Fixation

  Additional Materials (also see protocol 1)
  • Methanol: place 2 ml in each well of a 12‐well plate and precool methanol by leaving at −20°C overnight
  • PBS: precool to 4°C; distribute into four beakers and keep on ice
  • Phosphate‐buffered saline (PBS; see recipe) containing 2% (w/v) bovine serum albumin (BSA)
NOTE: See Table 12.34.1 to determine which autophagy‐related antibodies require methanol fixation.

Basic Protocol 2: Time‐Lapse Fluorescence Microscopy of Autophagosome Formation

  Materials
  • HEK‐293T cells stably expressing the autophagy markers GFP‐DFCP1 and mCherry‐Atg13
  • Complete medium (see recipe)
  • OptiMEM I reduced serum medium (Invitrogen, cat. no. 31985‐062)
  • X‐tremeGENE 9 DNA Transfection Reagent (Roche Applied Science, cat. no. 6365787001)
  • pECFP‐LC3: plasmid for the transient expression of CFP‐LC3 (supplied to our lab as a courtesy)
  • Amino acid starvation medium (see recipe)
  • Glass‐bottom culture dishes: Attofluor Cell Chamber (Invitrogen, cat. no. A‐7816)
  • Wide‐field microscope
  • Laboratory timer
  • Camera: Hamamatsu ORCA‐Flash4.0, pixel size 6.5 µm
  • Other components of wide‐field imaging system configured for time‐lapse fluorescence microscopy (see annotation to step 4 of this protocol)
  • ImageJ/Fiji software (download for free at http://fiji.sc/Fiji)
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Figures

Videos

Literature Cited

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Internet Resources
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