TITLE: Immunofluorescence microscopy of  R1441C  or VPS35 D620N MEF cells
AUTHORS: Chloe A Hecht, Herschel Dhekne, Wondwossen Yeshaw, Suzanne Pfeffer

[DESCRIPTION]
We present here a method to culture, fix, permeabilize, and stain R1441C or VPS35 D620N MEF cells to visualize transfected Myc-RILPL1 and endogenous TMEM55b or pRab10. 

[STEPS]
SECTION: Culturing, plating, and treating cells
1. Seed R1441C MEF cells or VPS35 D620N MEF cells at 50-60% confluency in a six well plate in 2 mL of complete DMEM (DMEM containing 10% FBS and 1% penicillin-streptomycin) 24 hours before transfection. 
SECTION: Culturing, plating, and treating cells
2. Transfect with Myc-RILPL1 plasmid with FuGENE 6 transfection reagent (E2691) at a 3:1 FuGENE:plasmid ratio using 2µg per well according to the manufacturer’s guidelines for 24 hours. 
SECTION: Culturing, plating, and treating cells
3. Trypsinize cells and plate onto 12 mm glass coverslips resting in a six well plate at 60% confluency and leave cells to attach to coverslips in a 37ºC incubator with 5% CO2 for 24 hours.
SECTION: Culturing, plating, and treating cells
4. If needed for MLi-2 conditions, add 200nM MLi-2 and leave in the incubator for 1.5 -2 hours. 
SECTION: Culturing, plating, and treating cells
5. For Nocodazole, add 20µM Nocodazole and leave in the incubator for 1.5-2 hours. 
SECTION: Fixing, permeabalizing, and mounting coverslips
6. Solutions needed: 
4% PFA in 1x PBS
0.2% Saponin in 1x PBS
2% BSA in 1x PBS (blocking solution)
SECTION: Fixing, permeabalizing, and mounting coverslips
7. Transfer each coverslip from the 6-well plate to a 24 well plate filled with ~400 µl 4% PFA per well and fix for 10 minutes at room temperature (all subsequent steps and solutions are at room temperature).
SECTION: Fixing, permeabalizing, and mounting coverslips
8. Wash 3x with 1x PBS. 
SECTION: Fixing, permeabalizing, and mounting coverslips
9. Permeabilize  cells with 0.2% Saponin in 1x PBS for 10 minutes.
SECTION: Fixing, permeabalizing, and mounting coverslips
10. Wash 3x in 1x PBS.
SECTION: Fixing, permeabalizing, and mounting coverslips
11. Block with 2% BSA in 1x PBS for 30 minutes.
SECTION: Fixing, permeabalizing, and mounting coverslips
12. Incubate primary antibody in blocking buffer for 2 hours.
SECTION: Fixing, permeabalizing, and mounting coverslips
12.1. Dilute antibodies (see Materials) as follows:
Rabbit anti-TMEM55B PA5-61760 (1:4000)
Rabbit anti-pRab10 (1:1000)
Mouse anti-Myc (1:1000)

SECTION: Fixing, permeabalizing, and mounting coverslips
13. Wash 3x with 1x PBS.
SECTION: Fixing, permeabalizing, and mounting coverslips
14. Incubate with 2º Antibody for 1 hour in the dark in blocking buffer. 
SECTION: Fixing, permeabalizing, and mounting coverslips
14.1. Dilute antibodies (see Materials) as follows:
Donkey anti-Rabbit 568 for TMEM55b and pRab10 (1:2000) and 
Donkey anti mouse 488 for Myc (1:2000)
DAPI (1:1000)

SECTION: Fixing, permeabalizing, and mounting coverslips
15. Wash 3x with 1x PBS.
SECTION: Fixing, permeabalizing, and mounting coverslips
16. Use a Kimwipe held on the edge of the coverslip to remove excess liquid; Invert the coverslip onto 3µl Mowiol on a Gold Seal glass slide (size 1x3’’, 1 mm thick).
SECTION: Fixing, permeabalizing, and mounting coverslips
17. Allow coverslips to dry at RT overnight.
SECTION: Fixing, permeabalizing, and mounting coverslips
18. Image