TITLE: Immunofluorescent Staining of phosphoRab10 in cultured cells
AUTHORS: Herschel  Dhekne, Suzanne R R Pfeffer

[DESCRIPTION]
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. 

[STEPS]
SECTION: Cell culture
1. 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.
SECTION: Cell culture
2. 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). 
SECTION: Cell culture
3. Plate approximately 30,000 cells on 12mm coverslips in 24 well plates submerged below 0.5 mL medium (~50% confluency at plating).

SECTION: Cell culture
3.1. Coverslips can be pre-treated with rat tail collagen. This helps A549 cells grow flatter, providing better organelle visualization.
SECTION: Cell culture
4. Cells may be visualized ~ 960 min after plating for immunofluorescence staining.

SECTION: Paraformaldehyde (PFA) fixation and blocking
5. Wash the cells 1X with 0.5 mL PBS.

SECTION: Paraformaldehyde (PFA) fixation and blocking
6. Fix the cells with 0.5 mL, 3% PFA in PBS for 30 min at Room temperature.

SECTION: Paraformaldehyde (PFA) fixation and blocking
7. Wash the cells 3X with 0.5 mL PBS per wash.

SECTION: Paraformaldehyde (PFA) fixation and blocking
8. For pRab10 staining, permeabilize the cells with 0.5 mL 0.2% Saponin for 5 min at Room temperature.

SECTION: Paraformaldehyde (PFA) fixation and blocking
8.1. Permeabilization with 0.1% Triton X-100 is also possible but yields lower sensitivity.
SECTION: Paraformaldehyde (PFA) fixation and blocking
9. Wash the cells 2X with PBS.
SECTION: Paraformaldehyde (PFA) fixation and blocking
10. After permeabilization, block the cells with 0.5 mL of 2% bovine serum albumin (BSA) in PBS for 30 min.

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

SECTION: Alternative fixation method: Methanol fixation and blocking
13. Incubate cells for 3 min- 5 min in a -20 °C freezer.

SECTION: Alternative fixation method: Methanol fixation and blocking
14. Aspirate methanol, wash cells twice with ice cold PBS.
SECTION: Alternative fixation method: Methanol fixation and blocking
15. Rehydrate cells slowly in PBS for 5 min on ice.

SECTION: Alternative fixation method: Methanol fixation and blocking
16. Antigen block with 2% BSA for 30 min (crucial to avoid background and artifacts).

SECTION: Alternative fixation method: Methanol fixation and blocking
16.1. No detergent permeabilization is needed as methanol solubilizes the lipids.
SECTION: Alternative fixation method: Methanol fixation and blocking
17. Anti-phospho-Rab10 antibody works OK using this fixation method in conjunction with PPM1H-KO A549 cells and MEFs
SECTION: Immunostaining
18. 
 

Primary antibody incubation: Rabbit anti-phosphoRab10 diluted to 0.5 μg/ml  in 2% BSA in PBS for  120 min.
SECTION: Immunostaining
18.1. Higher dilutions (0.25 μg/ml) work, but may decrease signal intensity.

SECTION: Immunostaining
19. After 120 min, wash cells 3X with PBS. 

SECTION: Immunostaining
20. Incubate the coverslips with a secondary goat anti-Rabbit Alexa-568 antibody (H+L, Invitrogen) diluted to 1 μg/ml in 2% BSA in PBS for45 min at Room temperature.

SECTION: Immunostaining
21. DAPI (Invitrogen) can be diluted 10,000X in the secondary antibody solution to co-stain the nucleus.
SECTION: Immunostaining
22. Wash the cells 3X with PBS. 
SECTION: Immunostaining
23. Mount the coverslips upside down by placement on 4 µL  Mowiol.