Jul 09, 2024

Public workspaceImmunofluorescence of ATP13A2 and CD63

DOI
dx.doi.org/10.17504/protocols.io.bp2l6xy3klqe/v1
  • 1Laboratory of Cellular Transport Systems, Department of Cellular and Molecular Medicine, KU Leuven, B-3000 Leuven, Belgium;
  • 2Aligning Science Across Parkinson’s (ASAP) Collaborative Research Network Chevy Chase, MD 20815, USA
Stephanie Vrijsen
Open access
DOI: dx.doi.org/10.17504/protocols.io.bp2l6xy3klqe/v1
Protocol Citation: Stephanie Vrijsen, Peter Vangheluwe 2024. Immunofluorescence of ATP13A2 and CD63. protocols.io https://dx.doi.org/10.17504/protocols.io.bp2l6xy3klqe/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 05, 2023
Last Modified: July 09, 2024
Protocol Integer ID: 91848
Keywords: ASAPCRN
Funders Acknowledgement:
Aligning Science Across Parkinson's (ASAP)
Grant ID: ASAP-000458
Abstract
This protocol describes the seeding, fixation and staining of samples for immunofluorescent visualization of ATP13A2 and CD63.
Materials
  • 4% paraformaldehyde (Thermo Fisher Scientific): J61899
  • BSA (Roth): 8076.4
  • Saponin (Sigma): S-2149
  • anti-ATP13A2 (Sigma): A3361
  • anti-CD63 (ExBio): 11-343-C100
  • alexa-488 goat anti-rabbit IgG (Invitrogen): R37116
  • alexa-594 goat anti-mouse IgG (Invitrogen): A11005
  • DAPI (Sigma): D9542
  • FluorSave (Merck): 345789-20ml
Seed cells: 75 000 cells/well (SH-SY5Y) in a 12-well plate with coverslips. Allow the cells to attach and grow for Duration48:00:00 .

2d
Wash the cells with ice-cold PBS.
Fix the cells with 4% paraformaldehyde Duration00:30:00 at 37 °C.

30m
Wash the cells twice with ice-cold PBS.
Permeabilization and blocking with a mixture of 5% BSA (Roth) and 0.5% saponin (Sigma) (diluted in PBS) (referred to as blocking buffer) for Duration01:00:00 on a shaker.

1h
Incubate with primary antibody (anti-ATP13A2, A3361, Sigma; anti-CD63, 11-343-C100, ExBio) (diluted 1/200 in blocking buffer) for Duration02:00:00 (40 µl antibody mixture is sufficient per coverslip in a wet chamber).

2h
Wash with DPBS for Duration00:05:00 on a shaker, repeat this step 3x.

5m
Incubate the samples in secondary antibody (alexa-488 goat anti-rabbit IgG R37116, Invitrogen; alexa-594 goat anti-mouse IgG, A11005, Invitrogen) (diluted 1/1000 in blocking buffer) for Duration00:30:00 on a shaker.

30m
Wash with DPBS for Duration00:05:00 on a shaker, repeat this step 3x.
5m
Incubate the samples in 200 ng/ml DAPI (D9542, Sigma) for Duration00:15:00 .

15m
Wash with DPBS for Duration00:05:00 on a shaker, repeat this step 3x.
5m
Mount the samples with FluorSave (345789-20ml, Merck).
Acquire images using a LSM880 microscope (Zeiss) with a 63x objective and Airyscan detector.