Mar 31, 2023
Immunofluorescence microscopy of R1441C or VPS35 D620N MEF cells
- Chloe A Hecht1,2,
- Herschel Dhekne1,2,
- Wondwossen Yeshaw1,2,
- Suzanne Pfeffer1,2
- 1Department of Biochemistry, Stanford University School of Medicine;
- 2Aligning Science Across Parkinson's Disease
Protocol Citation: Chloe A Hecht, Herschel Dhekne, Wondwossen Yeshaw, Suzanne Pfeffer 2023. Immunofluorescence microscopy of R1441C or VPS35 D620N MEF cells. protocols.io https://dx.doi.org/10.17504/protocols.io.j8nlkw1n5l5r/v1
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: March 21, 2023
Last Modified: March 31, 2023
Protocol Integer ID: 79201
Funders Acknowledgement:
Aligning Science Across Parkinson's Disease
Grant ID: 000463
Abstract
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.
Materials
Paraformaldehyde 16% (Electron Microscopy Sciences 50-980-487)
PBS (Cold Spring Harbor protocol)
TMEM55B polyclonal antibody (Proteintech 23992-1-AP)
Recombinant Anti-Rab10 (Phospho T73) antibody (Abcam ab241060)
Myc antibody (Biolegend 626802)
Donkey anti-Rabbit 568 Alexa Fluor (Thermo Fisher A10042)
Donkey anti-Mouse 488 Alexa Fluor (Thermo Fisher A-21202)
DMEM high glucose (Cytiva SH30243.01)
Fetal bovine serum (Sigma #F0926)
Microscope cover glass (Fisher 12-545-81)
FuGENE 6 transfection reagent (Promega E2691)
Culturing, plating, and treating cells
Culturing, plating, and treating 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.
Fixing, permeabalizing, and mounting coverslips
Fixing, permeabalizing, and mounting coverslips
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.
Image