Jun 16, 2026

Immunofluorescence and confocal microscopy

DOI
https://dx.doi.org/10.17504/protocols.io.4r3l21okxg1y/v1
  • Elisabeth Holzer1
  • 1Laboratory of Sascha Martens, Max Perutz Labs, University of Vienna, Austria
Elisabeth Holzer
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DOI: https://dx.doi.org/10.17504/protocols.io.4r3l21okxg1y/v1
Protocol Citation: Elisabeth Holzer 2026. Immunofluorescence and confocal microscopy. protocols.io https://dx.doi.org/10.17504/protocols.io.4r3l21okxg1y/v1
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
-
Created: July 31, 2025
Last Modified: June 16, 2026
Protocol  Integer ID: 223991
Keywords: ASAPCRN, confocal microscopy this protocol detail, confocal microscopy, immunofluorescence, microscopy
Funders Acknowledgements:
Aligning Science Across Parkinson’s (ASAP)
Grant ID: ASAP-000350
DOC Fellowship (Austrian Academy of Sciences)
Abstract
This protocol details the immunofluorescence and confocal microscopy.
Materials
Primary Antibodies

ABC
TargetDilutionSupplier/Details
ATG9A1:100CST, Cat# 13509; RRID:AB_2798241
ATG16L11:100CST, Cat# 8089; RRID:AB_10950320
EGFP1:100ChromoTek, Cat# ABIN398304; RRID:AB_10773374
LC3B1:100CST, Cat# 3868; RRID:AB_2137707
p62/SQSTM11:100Abnova, Cat# H00008878-M01; RRID:AB_437085
ULK11:100CST, Cat# 8054; RRID:AB_11178668
WIPI21:100Bio-Rad, Cat# MCA5780GA; RRID:AB_10845951

Secondary Antibodies

ABC
AntibodyDilutionSupplier/Details
AlexaFluor-488 goat anti-rat IgG (H+L)1:500Thermo Fisher, Cat# A-11008; RRID: AB_2338047
AlexaFluor-546 goat anti-mouse IgG (H+L)1:500Thermo Fisher, Cat# A-11003; RRID: AB_2534071
AlexaFluor-647 goat anti-rabbit IgG (H+L)1:500Thermo Fisher, Cat# A-21244; RRID: AB_2535812
Atg9A (D4O9D) Rabbit mAbCell Signaling TechnologyCatalog #13509
Atg16L1 (D6D5) Rabbit mAbCell Signaling TechnologyCatalog #8089
LC3B (D11) XP® Rabbit mAbCell Signaling TechnologyCatalog #3868
SQSTM1 monoclonal antibody (M01), clone 2C11AbnovaCatalog #H00008878-M01
ULK1 (D8H5) Rabbit mAbCell Signaling TechnologyCatalog #8054
WIPI2 antibody | 2A2Bio-Rad LaboratoriesCatalog #MCA5780GA
Goat anti-Rabbit IgG (H L) Cross-Adsorbed Secondary Antibody, Alexa Fluor™ 488Thermo Fisher ScientificCatalog #A-11008
Goat anti-Mouse IgG (H L) Cross-Adsorbed Secondary Antibody, Alexa Fluor™ 546Thermo Fisher ScientificCatalog #A-11003
Goat anti-Rabbit IgG (H L) Cross-Adsorbed Secondary Antibody, Alexa Fluor™ 647Thermo Fisher ScientificCatalog #A-21244
Lipofectamine™ 3000 Transfection ReagentThermo FisherCatalog #L3000008
Earle′s Balanced SaltsMerck MilliporeSigma (Sigma-Aldrich)Catalog #E3024
Bafilomycin A1 (CAS 88899-55-2)Santa Cruz BiotechnologyCatalog #sc-201550
VPS34-IN1ApexBio TechnologyCatalog # B6179
Triton™ X-100Merck MilliporeSigma (Sigma-Aldrich)Catalog #T8787
Bovine Serum AlbuminMerck MilliporeSigma (Sigma-Aldrich)Catalog #A9647
DAPI Fluoromount-G®Southern BiotechCatalog #0100-20
Equipment
12 mm #1.5 glass coverslips
NAME
Superior Marienfeld
BRAND
0117520
SKU
https://www.marienfeld-superior.com/cover-glasses-thickness-no-1-circular.html
LINK

Cell Seeding
Seed 100,000 cells per well on 12 mm #1.5 glass coverslips (Marienfeld, Cat# 0117520) in a suitable 6-well plate.
Incubate Overnight at 37 °C with 5% CO₂.
Treatment (Optional, As Indicated)
1d 2h
Treat cells for 02:00:00 with one of the following (as per figure legend):
  • EBSS (Sigma-Aldrich, Cat# E3024-500 mL ) + 100 nanomolar (nM) Bafilomycin A1 (Santa Cruz Biotechnology, Cat# sc-201550)
  • 2 micromolar (µM) VPS34-IN1 (ApexBio, Cat# APE-B6179)
1d 2h
Fixation
15m
Fix cells in cold methanol for 00:05:00 at -20 °C or 4% PFA for 00:10:00 at Room temperature .

15m
Wash cells with PBS.
Permeabilization and Blocking
1h
Incubate coverslips for 01:00:00 at Room temperature in blocking/permeabilization solution:
  • PBS
  • 0.25% (v/v) Triton‱ X-100 (Sigma-Aldrich, Cat# T8787)
  • 5% (v/v) BSA (Sigma-Aldrich, Cat# A9647)
1h
Antibody Staining
4h
Incubate cells with primary antibodies diluted in blocking buffer for 02:00:00 at Room temperature .
2h
Wash 3× with PBS.
Incubate with secondary antibodies (1:500 in blocking buffer) for 02:00:00 at Room temperature .
2h
Wash 3× with PBS.
Mounting
Mount coverslips onto glass microscopy slides using DAPI Fluoromount-G® (Southern Biotech, Cat# 0100-20).
Store mounted slides at -20 °C until imaging.
Imaging
Acquire confocal images using a Zeiss LSM900 with Airyscan 2 module and a Plan-Apochromat 63×/1.4 Oil DIC, WD 0.19 mm objective.
Process images using 2D Airyscan processing plug-in in Zeiss Zen Blue software.