Nov 07, 2025
Immunofluorescencent Staining of A-synuclein, TFEB, GCase and LAMP1 in treated cultured cells
- Sara Lucas Del-Pozo1,2,
- Giuseppe Uras1,2,3,
- Federico Fierli1,2,
- Veronica Lentini1,3,
- Sofia Koletsi1,2,
- Carlos Lazaro-Hernandez1,4,
- Kai-Yin Chau1,2,
- Derralynn A. Hughes5,
- Anthony H.V. Schapira1,2
- 1Department of Clinical and Movement Neurosciences, UCL Queen Square Institute of Neurology, London, UK;
- 2Aligning Science Across Parkinson's (ASAP) Collaborative Research Network, Chevy Chase, MD, USA;
- 3Department of Biomedical Science, University of Sassari, Viale San Pietro, Sassari 07100, Italy;
- 4Neurology Department, Vall d'Hebron University Hospital, Barcelona, Spain;
- 5Lysosomal Storage Disorders Unit, Royal Free Hospital NHS Foundation Trust and University College London, London, UK
Protocol Citation: Sara Lucas Del-Pozo, Giuseppe Uras, Federico Fierli, Veronica Lentini, Sofia Koletsi, Carlos Lazaro-Hernandez, Kai-Yin Chau, Derralynn A. Hughes, Anthony H.V. Schapira 2025. Immunofluorescencent Staining of A-synuclein, TFEB, GCase and LAMP1 in treated cultured cells. protocols.io https://dx.doi.org/10.17504/protocols.io.36wgqpk7kvk5/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: November 02, 2023
Last Modified: November 07, 2025
Protocol Integer ID: 231666
Keywords: ASAPCRN, synuclein protein, synuclein, lamp1 by confocal immunofluorescence, mutant form of the snca gene, differentiated cell, immunofluorescencent, snca gene, sy5y parental cell line, mutant form, confocal immunofluorescence, cultured cells this protocol
Funders Acknowledgements:
Aligning Science Across Parkinson's
Grant ID: ASAP-000420
Abstract
This protocol can be used to detect the amount and localization of endogenous a-synuclein, TFEB, GCase and LAMP1 by confocal immunofluorescence. In our hands, we performed the same protocol in undifferentiated SH-SHY5Y parental cell line and in SH-SY5Y expressing a mutant form of the SNCA gene which leads to a rapid aggregation of a-synuclein protein following its interaction with lipid-membrane organelles. In this mutant model, the familiar PD mutation E46K was “amplified” by introducing two analogous extra-lysine mutations into the nearby KTKEGV repetitive motive in positions E35K, E46K, and E61K. Subsequently, we applied a differentiation protocol both to the SH-SY5Y parental cell line and the 3K-SNCA overexpressing ones. In differentiated cells, the same immunostaining protocol was again conducted.
Materials
Cell lines:
SH-SY5Y cells were used as parental control cells (#91396-88-2, Atcc,UK). SH-SY5Y cells constitutively expressing the 3K-SNCA gene were a kind gift from Dr. Ulf Dettmer laboratory (Harvard, USA).
Compounds:
Compounds YM201636 (#13576, Cayman Chemical, Ann Arbour, US) and ambroxol hydrochloride (#A9797, Merck, London, UK) were diluted in DMSO to obtain a 10mM stock solution. Subsequent dilutions were all made in sterile PBS (Gibco® REF 14190-144) and stored at -80C until usage.
DMEM, high glucose, GlutaMAX™ SupplementThermo FisherCatalog #61965026
Neurobasal Medium (1x)Gibco - Thermo Fisher ScientificCatalog #21103049
Retinoic acid, 97%Thermo Fisher ScientificCatalog #044540.04
B-27™ Supplement (50X), serum freeGibco - Thermo Fisher ScientificCatalog #17504044
GlutamaxGibco - Thermo Fisher ScientificCatalog #35050-061 Human Brain-Derived Neurotrophic Factor (BDNF)Cell Signaling TechnologyCatalog #3897
Geltrex™ LDEV-Free Reduced Growth Factor Basement Membrane MatrixThermo FisherCatalog #A1413202
DMEM/F-12, GlutaMAX™ SupplementThermo FisherCatalog #31331028
YM-201636Cayman Chemical CompanyCatalog #13576
Ambroxol hydrochlorideMerck MilliporeSigma (Sigma-Aldrich)Catalog #A9797
Phosphate buffered saline (PBS) without Ca/Mg Thermo Fisher ScientificCatalog #14190144
DMEM/F-12, GlutaMAX™ SupplementThermo FisherCatalog #31331028
Fetal Bovine SerumGibco - Thermo Fisher ScientificCatalog #10270106
Penicillin-StreptomycinGibco - Thermo Fisher ScientificCatalog #15140122
Trypsin (2.5%), no phenol redThermo FisherCatalog #15090046
Versene SolutionThermo FisherCatalog #15040066
Normal Goat SerumAbcamCatalog #ab7481
Recombinant Anti-Alpha-synuclein antibodyAbcamCatalog #ab138501
TFEB (D2O7D) Rabbit mAbCell Signaling TechnologyCatalog #37785
Purified Mouse Anti-Human Lamp-1BD BiosciencesCatalog #611042
Anti-Alpha-synuclein antibody [4D6] - BSA and Azide freeAbcamCatalog #ab1903
F(ab)2-Goat anti-Mouse IgG (H L) Cross-Adsorbed Secondary Antibody, Alexa Fluor™ 488Invitrogen - Thermo FisherCatalog #A-11017
Goat anti-Rabbit IgG (H L) Cross-Adsorbed Secondary Antibody, Alexa Fluor™ 568Invitrogen - Thermo FisherCatalog #A-11011
4,6-Diamidino-2-Phenylindole, Dihydrochloride (DAPI)Thermo Fisher ScientificCatalog #D1306
Cell differentiation protocol
Differentiate the wild-type and 3K-SNCA overexpressing SH-SY5Y into neurons using retinoic acid-based protocol.
Detach the 70% confluent non-differentiated cells using trypsin and Versene® (Gibco® REF 61965-026) and resuspend in Neurobasal media (Gibco® REF 21103-049) supplemented with 30M retinoic acid (ThermoFisher® # 044540.04), 1X vitamin B-27 (Gibco® REF 17504-044), 1X Glutamax® (Gibco® REF 35050-061), and 5ng/ml brain-derived neurotrophic factor (BDNF) (Cell Signaling® REF 3897S).
Replace the differentiation medium every 48 hours, with a neuronal morphology appearing after 4 days of culture.
Use the differentiated neurons for downstream analysis after 10 days in culture.
Seed the cells in Geltrex® (Gibco® REF A1413202) Matrix solution diluted in DMEM/F-12 medium (Gibco® REF 31331-028) (1:100) coated plates at 1.5 × 105 cells/ml.
Cell culture of non-differentiated cells
Culture the SH-SY5Y cells and 3K-SNCA overexpressing cells in 1:1 DMEM/F12 media (Gibco® REF 31331-028) with Glutamax® (Gibco®, REF 35050-061) supplemented with 10% fetal bovine serum (FBS) (Gibco® REF 10270-106) and 1X Penicillin/Streptomycin (Gibco® REF 15140-122). Passeage the cells appropriately to maintain a confluency between 50-70%.
Subsequently, detach the cells using Trypsin 10x (Gibco® REF 15090-046) into Versene 1X (Gibco® REF 15040-066) (1:1), count, and seed in a new 96-well plate. In order to acquire images with the Opera Phenix® Plus High-Content Screening System, seed the cells in a specific type of 96 well plate, the Phenoplate®-96 TC (PerkinElmer®, #39269097).
Seed the 3*104 cells into each well, avoiding placing them into borders, rows and columns to prevent plate-edge effects.
24 hours post seeding, discard the medium and replace it with 100 µL of medium containing the appropriate compound or vehicle.
Left the cells to incubate for 24 hours and subsequently use it for downstream analysis.
Cell culture of differentiated cells
1d
After 10 days in the cell culture described above (see Cell differentiation protocol section), use the differentiated neurons for downstream analysis.
Detach the differentiated cells using Trypsin 10x (Gibco® REF 15090-046) into Versene 1X (Gibco® REF 15040-066) (1:1), count, and seed in a new 96-well plate. In order to acquire images with the Opera Phenix® Plus High-Content Screening System, seed the cells in a specific type of 96 well plate, the Phenoplate®-96 TC (PerkinElmer®, #39269097).
Seed the 3*104 cells into each well, avoiding placing them into borders, rows and columns to prevent plate-edge effects.
24 hours post seeding, discard the medium and replace it with 100 µL of differentiation medium (see Cell differentiation protocol section) containing the appropriate compound or vehicle.
Left the cells to incubate for 24:00:00 and subsequently used for downstream analysis.
1d
Cell Staining
4h 50m
Following compound treatment for 24 hours, remove the cell culture medium and fix the cells with 100 µL of 4% para-formaldehyde (PFA) (Severn Biotech Ltd. REF 40-7401-05) for 00:20:00 at Room temperature .
20m
Subsequently, remove the PFA and add the 100 µL of ice-cold methanol to each well for 00:20:00 .
20m
Discard the methanol and was each well 3 times with 1X PBS (Gibco® REF 14190-144).
Block the cells with 100 µL 5% normal goat serum (NGS) (ab7481, Abcam®, Cambridge, UK) for 01:00:00 at Room temperature .
1h
Add the primary antibodies according to the experiment: (i) monoclonal rabbit anti-synuclein 1:750 (#ab138501, Abcam®, Cambridge UK), (ii) monoclonal mouse anti-GBA 1:1000 (Anti-GBA mAb (2E2), #AP1140-100UG, Calbiochem®), (iii) monoclonal rabbit anti-TFEB 1:500 (Anti-TFEB D207D Rabbit mAb #37785 Cell Signalling®), (iv) monoclonal mouse anti-LAMP1 1:1000 (#611042, BD® Transduction Lab), (v) monoclonal mouse anti-synclein 1:750 (#ab1903, Abcam®, Cambridge, UK).
Upon addition of primary antibody, left the cells to incubate Overnight at 4 °C .
1h
Wash the cells 3 times with PBS and incubated for 02:00:00 at Room temperature with secondary antibody according to the experiment: AlexaFluor 488 1:2000 (Invitrogen® #A11017), AlexaFluor 568 1:2000 (Invitrogen® #A11011), AlexaFluor 647 1:2000 (Invitrogen® #A21244).
2h
Subsequently, wash the cells 3 times with 1X PBS, and incubate them for 00:10:00 with 100 µL of DAPI (Invitrogen® #D1306) solution (5 µL ).
10m
Wash each well 3 times with 1X PBS and fill with 200 µL of PBS. Store the plates at 4 °C avoiding light until analysis.
Image acquisition
Perform the cell imaging using the Opera Phenix® Plus High-Content Screening System (PerkinElmer®, Waltham US) using confocal mode. Acquire each channel separately to avoid overlap signal.
Acquire DAPI with laser power at 30% with exposure set at 100ms.
Acquire 488 channel with laser power at 70% with exposure at 100ms.
Acquire 564 channel acquired with laser power at 70% with exposure at 100ms.
Acquire 647 channel with a laser power at 80% with exposure at 100ms.
Acquire z-stack of 2 um for each image.