Immunofluorescent Staining of phosphoRab10 in cultured cells
Suzanne R Pfeffer, Herschel Dhekne
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.
Attachments
Steps
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 0.5mL 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 ~ 16h 0m 0s after plating for immunofluorescence staining.
Paraformaldehyde (PFA) fixation and blocking
Wash the cells 1X with 0.5mL PBS.
Fix the cells with 0.5mL, 3% PFA in PBS for 0h 30m 0s at Room temperature.
Wash the cells 3X with 0.5mL PBS per wash.
For pRab10 staining, permeabilize the cells with 0.5mL 0.2% Saponin for 0h 5m 0s at Room temperature.
Permeabilization with 0.1% Triton X-100 is also possible but yields lower sensitivity.
Wash the cells 2X with PBS.
After permeabilization, block the cells with 0.5mL of 2% bovine serum albumin (BSA) in PBS for 0h 30m 0s.
Alternative fixation method: Methanol fixation and blocking
Methanol fixation is needed to stain microtubule-based structures (centrioles).
Fix cells by gently adding -20°C methanol to coverslips.
Incubate cells for 0h 3m 0s- 0h 5m 0s in a -20°C freezer.
Aspirate methanol, wash cells twice with ice cold PBS.
Rehydrate cells slowly in PBS for 0h 5m 0s On ice.
Antigen block with 2% BSA for 0h 30m 0s (crucial to avoid background and artifacts).
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
Primary antibody incubation: Rabbit anti-phosphoRab10 diluted to 0.5μg/ml in 2% BSA in PBS for 2h 0m 0s.
Higher dilutions (0.25μg/ml) work, but may decrease signal intensity.
After 2h 0m 0s, wash cells 3X with PBS.
Incubate the coverslips with a secondary goat anti-Rabbit Alexa-568 antibody (H+L, Invitrogen) diluted to 1μg/ml in 2% BSA in PBS for0h 45m 0s at Room temperature.
DAPI (Invitrogen) can be diluted 10,000X in the secondary antibody solution to co-stain the nucleus.
Wash the cells 3X with PBS.
Mount the coverslips upside down by placement on 4µL Mowiol.
