May 27, 2025

Immunocytochemistry of autophagosome-associated proteins

DOI
https://dx.doi.org/10.17504/protocols.io.rm7vzqpb8vx1/v1
  • Sierra Palumbos1,
  • Erika Holzbaur1
  • 1University of Pennsylvania
Sierra Palumbos
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DOI: https://dx.doi.org/10.17504/protocols.io.rm7vzqpb8vx1/v1
Protocol Citation: Sierra Palumbos, Erika Holzbaur 2025. Immunocytochemistry of autophagosome-associated proteins. protocols.io https://dx.doi.org/10.17504/protocols.io.rm7vzqpb8vx1/v1
Manuscript citation:
https://doi.org/10.1101/2024.11.07.621551
License: This is an open access  protocol  distributed under the terms of the  Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: May 27, 2025
Last Modified: May 27, 2025
Protocol  Integer ID: 219002
Keywords: immunocytochemistry of autophagosome, autophagosome associtated protein, autophagosome, proteins protocol for immunocytochemistry, associated proteins protocol, associtated protein, primary cortical neuron, immunocytochemistry, protein, antibody, neuron, other cultured cell type, cortical neuron
Funders Acknowledgements:
National Institute of Neurological Disorders and Stroke
Grant ID: R01-NS060698
National Institute of Neurological Disorders and Stroke
Grant ID: F32-NS129586
Michael J. Fox Foundation for Parkinson's Research (MJFF)
Grant ID: MJFF-021130
Michael J. Fox Foundation for Parkinson's Research (MJFF)
Grant ID: MJFF-15100
Michael J. Fox Foundation for Parkinson's Research (MJFF)
Grant ID: MJFF-019411
Abstract
Protocol for immunocytochemistry used on primary cortical neurons in Palumbos et al., 2025. Protocol can be adapted to other cultured cell types and antibodies. Described protocol works for autophagosome associtated proteins
Fix Cells
Heat 4% paraformaldehyde (PFA) to 37 °C

Add 4% sucrose by volume
Wash cultured cells 2X with warmed 1X PBS

Fix with 4% PFA + sucrose for 00:08:00 at 37 °C

Wash with 1X PBS for 5 minutes
Repeat wash 2X

Stopping point - store at 4°C in 1X PBS or continue
Permeabilize Cells
Add permeabilizing agent and incubate cells.
Note
Permeabilization method should be determined based on protein(s) to be detected.
Follow Step 8.2 (MeOH permeabilization) for autophagosome or lysosome associated proteins
Follow Step 8.3 (Triton-X permeabilization) for other visualization
*You may need to try both methods to find best fit for a given antibody

MeOH permeabilization - Add 1 mL cold (-20°C) pure MeOH to well and place in -20°C for 8 minutes.
Triton X permeabilization - Add 0.2% Triton X-100 in PBS to well for 15 minutes at room temperature (not ideal for autophagosomes or lysosomes).
Wash 3X for 5 minutes with 1X PBS.
Block Cells
Prepare block (5% goat serum, 1% BSA, + sodium azide) in advance. Filter sterilize and keep at 4°C.
Add block to plates and incubate for 01:00:00 at Room temperature .

Primary Antibody Incubation
Prepare primary antibody dilution in block (1:250 good starting dilution if unknown).
Incubate overnight with primary antibody at 4°C.
Secondary Antibody Incubation
Wash 3X for 5 minutes with 1X PBS.
Prepare secondary antibody dilution in block (1:1000 good starting dilution).
Incubate at Room temperature for 02:00:00 .

Wash 3X for 5 minutes with 1X PBS.
Add Hoechst 1:1000 for 10 minutes.
Wash 3X for 5 minutes with 1X PBS.
Mount coverslip for imaging.