Apr 15, 2026

Immunocytochemistry - subcellular localization of ATP13A4

DOI
https://dx.doi.org/10.17504/protocols.io.kxygxwy4kv8j/v1
  • 1KU Leuven;
  • 2Aligning Science Across Parkinson's (ASAP) Collaborative Research Network, Chevy Chase, MD 20815
Sarah van Veen
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DOI: https://dx.doi.org/10.17504/protocols.io.kxygxwy4kv8j/v1
Protocol Citation: Sarah van Veen, Peter Vangheluwe 2026. Immunocytochemistry - subcellular localization of ATP13A4. protocols.io https://dx.doi.org/10.17504/protocols.io.kxygxwy4kv8j/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
We use this protocol and it's working
Created: December 11, 2024
Last Modified: April 15, 2026
Protocol  Integer ID: 115008
Keywords: ASAPCRN, ICC, ATP13A4, subcellular localization of atp13a4, atp13a4 in hela cell, atp13a4, subcellular localization, hela cell, immunocytochemistry, cell, using immunocytochemistry
Funders Acknowledgements:
Aligning Science Across Parkinson's
Grant ID: ASAP-000458
Fonds Wetenschappelijk Onderzoek (FWO)
Grant ID: G011424N
Abstract
This protocol describes an optimized method for visualizing the subcellular localization of over-expressed ATP13A4 in HeLa cells using immunocytochemistry.

Materials
PBS-T:
  • PBS
  • 0.5% Tween-20

Materials
  • PEI MAXTM (Cat# 24765-100, Polysciences)
  • Paraformaldehyde, 4% in PBS (Cat# J61899.AP, Thermo Fisher)
  • Triton X-100 (Cat# 93443, Sigma-Aldrich)
  • Dulbecco's Phosphate Buffered Saline without calcium chloride and magnesium chloride (DPBS) (Cat# 14190144, GIBCO)
  • Tween-20 (Cat# A4974.0500, PanReac AppliChem)
  • Glycine (Cat# G8898, Sigma-Aldrich)
  • FBS
  • Bovine Serum Albumin (BSA) Fraction V (Cat# 8076.4, Carl Roth)
  • EEA1 (Cat# 610457, RRID:AB_397830, BD Biosciences)
  • TOMM22 (Cat# HPA003037, RRID:AB_1080329, Sigma-Aldrich)
  • CD63 (Cat# 11-343-C025, RRID:AB_10755612, EXBIO Praha)
  • LAMP1 (Cat# 9091, RRID:AB_2687579, Cell Signaling Technology)
  • DAPI (Cat# 62248, Thermo Fisher) 
  • Goat anti-Rabbit IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor‱ 488 (Cat# A-11008, RRID:AB_143165, Thermo Fisher Scientific)
  • Alexa-488 goat anti-mouse IgG (Cat# A-11017, RRID:AB_2534084, Thermo Fisher Scientific)
  • FluorSave‱ Reagent (Cat# 345789, Millipore)
Safety warnings
Follow institutional guidelines for the disposal of biological and chemical waste.
Cell transfection
Seed HeLa cells into a 15 cm dish at a density appropriate to achieve 70–80% confluence by the following day. Allow the cells to attach and grow for approximately 24:00:00 .
1d
The following day, transfect the cells with pcDNA3.1-mCherry-msAtp13a4 using PEImax.
In a sterile tube, dilute 12 µg plasmid DNA (pcDNA3.1-mCherry-msAtp13a4) in 2.5 mL serum-free medium.
Add 48 µL PEI MAXTM (1ug/uL) to the diluted DNA. Mix immediately by vortexing or pipeting.
Note
The volume of PEI used is based on a 4:1 ratio of PEI (ug):total DNA (ug).


Incubate 00:15:00 at Room temperature .
15m
Add the DNA/PEI mixture dropwise to the cells and incubate under standard culture conditions for 24:00:00 .
1d
The following day, trypsinize the transfected cells and plate onto cover slips. Allow the cells to attach and grow for approximately 24:00:00 .
1d
Antibody staining
2d 5h 55m
Fix cells with 4% PFA in PBS for 00:30:00 at 37 °C .
30m
Wash cells 3 times in PBS-T (00:05:00 each wash) at Room temperature on a rocker.
5m
Permeabilize cells by incubating in 0.5% Triton X-100 solution for 00:30:00 at Room temperature on a rocker.

30m
Wash cells 3 times in PBS-T (00:05:00 each wash) at Room temperature on a rocker.
5m
Incubate in 0.1 M glycine in PBS for 01:00:00 at Room temperature on a rocker.
1h
Wash cells 3 times in PBS-T (00:05:00 each wash) at Room temperature on a rocker.
5m
Block cells in PBS-T supplemented with 10% FBS and 1% BSA for 01:00:00 at Room temperature on a rocker.

1h
Wash cells 3 times in PBS-T (00:05:00 each wash) at Room temperature on a rocker.
5m
Incubate cells Overnight at 4 °C with primary antibodies (anti-EEA1, anti-CD63, anti-LAMP1, anti-TOMM22) diluted 1:200 in PBS-T containing 1% FBS and 0.1% BSA on a rocker.
1h
The next day, wash cells 3 times in PBS-T (00:05:00 each wash) on a rocker.
5m
Block cells in PBS-T supplemented with 10% FBS and 1% BSA for 00:30:00 at Room temperature on a rocker.
30m
Wash cells 3 times in PBS-T (00:05:00 each wash) at Room temperature on a rocker.
5m
Incubate cells with Alexa Fluor 488-conjugated secondary antibody (1:1000 in PBS-T with 1% FBS and 0.1% BSA) for 00:30:00 at Room temperature , in the dark on a rocker.
30m
Wash cells 3 times in PBS-T (00:05:00 each wash) on a rocker.
5m
Incubate cells with DAPI (1:5000 in PBS-T) for 00:15:00 at Room temperature , protected from light, on a rocker.
15m
Wash cells 3 times in PBS-T (00:05:00 each wash) on a rocker, protected from light.
5m
Wash cells Overnight with PBS-T at 4 °C on a rocker, protected from light.

1d
Mount coverslips onto glass slides using mounting medium (FluorSave‱ Reagent).
Allow the slides to set Overnight at Room temperature .
1d
Imaging
2d 5h 55m
Acquire images using a Zeiss LSM780 confocal microscope or equivalent at 63x magnification.