Jun 16, 2022

Public workspaceImmunofluorescent Staining of phosphoRab10 in cultured cells

DOI
dx.doi.org/10.17504/protocols.io.ewov1nmzkgr2/v1
  • 1Department of Biochemistry, Stanford University School of Medicine
Suzanne R Pfeffer
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DOI: dx.doi.org/10.17504/protocols.io.ewov1nmzkgr2/v1
External link: https://doi.org/10.1042/BCJ20220308
Protocol Citation: Herschel Dhekne, Suzanne R Pfeffer 2022. Immunofluorescent Staining of phosphoRab10 in cultured cells. protocols.io https://dx.doi.org/10.17504/protocols.io.ewov1nmzkgr2/v1
Manuscript citation:
Malik AU, Karapetsas A, Nirujogi RS, Chatterjee D, Phung TK, Wightman M, Gourlay R, Morrice N, Mathea S, Knapp S, Alessi DR, PKC isoforms activate LRRK1 kinase by phosphorylating conserved residues (Ser1064, Ser1074 and Thr1075) within the COR GTPase domain. Biochemical Journal 479(18). doi: 10.1042/BCJ20220308
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: June 08, 2022
Last Modified: May 31, 2024
Protocol Integer ID: 64193
Keywords: Immunofluorescent Staining, PhosphoRab10, Cultured cell, ASAPCRN
Funders Acknowledgement:
Aligning Science Across Parkinson's Disease
Grant ID: Team Alessi
Abstract
This protocol can be used to detect the amount and localization of endogenous phospho-Rab10 by light microscopy. Cells that yield detectable, endogenous phosphorylated Rab10 without the need to express LRRK2 include: Mouse embryonic fibroblasts (MEFs; wild type and LRRK2 R1441C or G2019S, VPS35 D620N); A549 PPM1H knock-out; NIH-3T3; immunopanned primary rat astrocytes. In our hands, HeLa, hTert-RPE, A549, HEK-293T, and ShSy5y cells can be immunostained for phosphorylated Rab10 but require exogenous expression of wildtype or pathogenic mutant LRRK2 or addition of pharmacological agents. Cells should be Mycoplasma free.
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Materials
Materials

  • 24 well plastic tissue culture plates
  • Collagen coated 12mm coverslips
  • Cells: MEFs (WT/R1441C/VPS35 D260N), PPM1H-KO A549
  • ReagentMEM Non-Essential Amino Acids Solution (100X)Thermo Fisher ScientificCatalog #11140050
  • ReagentFetal Bovine SerumAtlanta BiologicalsCatalog #S11550
  • ReagentRecombinant Anti-RAB10 (phospho T73) antibody [MJF-R21-22-5] (ab241060)AbcamCatalog #ab241060
  • Donkey anti-Rabbit-Alexa 568 highly cross-adsorbed H+L (Life Technologies)
  • Paraformaldehyde (PFA, Sigma)
  • Triton X-100 (Sigma)
  • Saponin (Sigma)
  • 2% BSA in PBS
  • Methanol (Sigma) stored at Temperature-20 °C




Cell culture
Cell culture
Culture the cells in high glucose DMEM medium with glutamine and sodium pyruvate, 10% fetal bovine serum, with additional non-essential amino acids and Penicillin/Streptomycin.
MEFs are generally flat and occupy a relatively large surface area: cell counts per confluent dish are ~5X lower than other common cell lines (eg. HeLa).
Plate approximately 30,000 cells on 12mm coverslips in 24 well plates submerged below Amount0.5 mL medium (~50% confluency at plating).

Coverslips can be pre-treated with rat tail collagen. This helps A549 cells grow flatter, providing better organelle visualization.
Cells may be visualized ~ Duration16:00:00 after plating for immunofluorescence staining.

16h
Paraformaldehyde (PFA) fixation and blocking
Paraformaldehyde (PFA) fixation and blocking
Wash the cells 1X with Amount0.5 mL PBS.

Wash
Fix the cells with Amount0.5 mL , 3% PFA in PBS for Duration00:30:00 at TemperatureRoom temperature .

30m
Wash the cells 3X with Amount0.5 mL PBS per wash.

Wash
For pRab10 staining, permeabilize the cells with Amount0.5 mL 0.2% Saponin for Duration00:05:00 at TemperatureRoom temperature .

5m
Permeabilization with 0.1% Triton X-100 is also possible but yields lower sensitivity.
Wash the cells 2X with PBS.
Wash
After permeabilization, block the cells with Amount0.5 mL of 2% bovine serum albumin (BSA) in PBS for Duration00:30:00 .

30m
Alternative fixation method: Methanol fixation and blocking
Alternative fixation method: Methanol fixation and blocking
Methanol fixation is needed to stain microtubule-based structures (centrioles).
Fix cells by gently adding Temperature-20 °C methanol to coverslips.

Pipetting
Incubate cells for Duration00:03:00 - Duration00:05:00 in a Temperature-20 °C freezer.

8m
Incubation
Aspirate methanol, wash cells twice with ice cold PBS.
Wash
Rehydrate cells slowly in PBS for Duration00:05:00 TemperatureOn ice .

5m
Antigen block with 2% BSA for Duration00:30:00 (crucial to avoid background and artifacts).

30m
No detergent permeabilization is needed as methanol solubilizes the lipids.
Anti-phospho-Rab10 antibody works OK using this fixation method in conjunction with PPM1H-KO A549 cells and MEFs
Immunostaining
Immunostaining
4h 45m
4h 45m

Note
Staining can be carried out following blocking after either PFA or Methanol fixation.

Primary antibody incubation: Rabbit anti-phosphoRab10 diluted to Concentration0.5 μg/ml in 2% BSA in PBS for Duration02:00:00 .
2h
Higher dilutions (Concentration0.25 μg/ml ) work, but may decrease signal intensity.

After Duration02:00:00 , wash cells 3X with PBS.

2h
Wash
Incubate the coverslips with a secondary goat anti-Rabbit Alexa-568 antibody (H+L, Invitrogen) diluted to Concentration1 μg/ml in 2% BSA in PBS forDuration00:45:00 at TemperatureRoom temperature .

45m
Incubation
DAPI (Invitrogen) can be diluted 10,000X in the secondary antibody solution to co-stain the nucleus.
Wash the cells 3X with PBS.
Wash
Mount the coverslips upside down by placement on Amount4 µL Mowiol.