Immunofluorescence microscopy of R1441C or VPS35 D620N MEF cells

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.

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.

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.

If needed for MLi-2 conditions, add 200nM MLi-2 and leave in the incubator for 1.5 -2 hours.

For Nocodazole, add 20µM Nocodazole and leave in the incubator for 1.5-2 hours.

Solutions needed:

• 4% PFA in 1x PBS
• 0.2% Saponin in 1x PBS
• 2% BSA in 1x PBS (blocking solution)

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).

Wash 3x with 1x PBS.

Permeabilize cells with 0.2% Saponin in 1x PBS for 10 minutes.

Wash 3x in 1x PBS.

Block with 2% BSA in 1x PBS for 30 minutes.

Incubate primary antibody in blocking buffer for 2 hours.

Dilute antibodies (see Materials) as follows:

Rabbit anti-TMEM55B PA5-61760 (1:4000)

Rabbit anti-pRab10 (1:1000)

Mouse anti-Myc (1:1000)

Wash 3x with 1x PBS.

Incubate with 2º Antibody for 1 hour in the dark in blocking buffer.

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)

Wash 3x with 1x PBS.

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).

Allow coverslips to dry at RT overnight.

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