Apr 01, 2022
Version 2
Immunofluorescence of RAB5 and FLAG-EEA1 puncta after Dynamin-1 and -2 inhibition with Dyngo4a V.2
- Hankum Park1,2,3,
- Frances V Hundley1,2,
- Harper JW1,2
- 1Department of Cell Biology, Harvard Medical School Boston, MA 02115, USA;
- 2Aligning Science Across Parkinson's (ASAP) Collaborative Research Network, Chevy Chase, MD 20815, USA;
- 3Current affiliation: Seoul National University, School of Dentistry
Protocol Citation: Hankum Park, Frances V Hundley, Harper JW 2022. Immunofluorescence of RAB5 and FLAG-EEA1 puncta after Dynamin-1 and -2 inhibition with Dyngo4a. protocols.io https://dx.doi.org/10.17504/protocols.io.ewov146xkvr2/v2Version created by Frances V Hundley
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 25, 2022
Last Modified: May 31, 2024
Protocol Integer ID: 59922
Keywords: endosome, immunoflourescence, EEA1, RAB5, ASAPCRN
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Abstract
Selective purification of early endosomes can be achieved through affinity capture of the early endosome-associated protein EEA1 (termed Endo-IP) (Park et al. 2022). These purified endosomes can be used for proteomic and lipidomic studies to obtain snapshots of early endosomes. Here, we present an immunofluorescence protocol to assess the extent of colocalization between FLAG-EEA1 and RAB5 with and without the Dynamin-1 and -2 (DNM1/2) inhibitor Dyngo4a.
Materials
| A | B | C | |
| REAGENT or RESOURCE | SOURCE | IDENTIFIER | |
| Antibodies | |||
| anti-RAB5 (C8B1) rabbit mAb | Cell Signaling Technology | 3547 | |
| anti-DYKDDDDK tag, mouse mAb (FG4R) | Thermo Fisher Scientific | MA1-91878 | |
| Alexa Fluor 594 Goat anti-Rabbit IgG (H+L) cross-adsorbed secondary antibody | Thermo Fisher Scientific | A-11012 | |
| Alexa Fluor 488 Goat anti-Mouse IgG (H+L) highly cross-adsorbed secondary antibody | Thermo Fisher Scientific | A-11029 | |
| Chemicals, peptides, and recombinant proteins | |||
| Hoechst 33342, Trihydrochloride, Trihydrate | Thermo Fisher Scientific | H3570 | |
| Poly-l-lysine solution, 0.01% | Sigma-Aldrich | P4832 | |
| Paraformaldehyde, 16% solution | Electron Microscopy Services | 15710 | |
| ProLong Glass Antifade Mountant | Thermo Fisher Scientific | P36982 | |
| Dyngo4a | Cayman Chemical | 29479 | |
| Software and algorithms | |||
| Fiji | ImageJ and SciJava projects | https://imagej.net/software/fiji/ | |
| MetaMorph v7.10 | Molecular Devices | https://www.moleculardevices.com/products/cellular-imaging-systems/acquisition-and-analysis-software/metamorph-microscopy#gref |
Preparation of coverslips
Preparation of coverslips
45m
45m
Coat No.1.5 coverslips in 0.01% poly-L-lysine solution. Incubate at 37 °C for 00:15:00
15m
Aspirate poly-L-lysine solution and wash coverslips three times with sterile DPBS.
Dry coverslips at 37 °C for 00:15:00 .
15m
Seed cells
Seed cells
15m
15m
Split 293EL cells expressing 3XFLAG-EEA1 (see protocol dx.doi.org/10.17504/protocols.io.byi7puhn) by standard methods and seed onto the prepared coverslips such that they will be approximately 70% confluent the next day.
Dyngo4a treatment
Dyngo4a treatment
3h
3h
The next day, check that cells are approximately 70% confluent.
Aliquot and warm serum-free DMEM to 37 °C .
Dilute DMSO (for control) and Dyngo4a (treatment) into warmed serum-free DMEM to a final concentration of 0.4% DMSO and 20 micromolar (µM) Dyngo4a. Note: if using 5 millimolar (mM) Dyngo4a stocks in DMSO, the final concentration DMSO in both control and treated samples will be 0.4%.
Note
Protect Dyngo4a from light, and thaw just before use.
Note
The exact dose of Dyngo4a and length of treatment will vary by cell line.
Aspirate existing media from cells growing on coverslips, and add new media containing either DMSO or Dyngo4a. Return cells to incubator for 03:00:00
3h
After treatment, neutralize Dyngo4a by aspirating DMSO or Dyngo4a media and washing cells once with DMEM with 10% serum and 0.4% DMSO. Wash cells once with DPBS.
Sample fixation and staining
Sample fixation and staining
1d
1d
Fix cells in 4% paraformaldehyde solution in DPBS for 00:15:00 at 25 °C .
15m
Wash samples three times in DPBS. Block samples for 01:00:00 at 25 °C in blocking buffer (1% BSA, 0.15% Triton X-100, DPBS).
1h
Remove blocking solution, and incubate samples in primary antibody solution [anti-RAB5 (Cell Signaling Technology, 3547) at 1:200 and anti-DYKDDDDK (Thermo Fisher Scientific, MA1-91878, which detects the FLAG epitope) at 1:200 in blocking solution] Overnight at 4 °C . Include single primary antibody controls and no primary antibody controls.
The next day, remove the primary antibody solution, wash samples three times with blocking solution, and incubate in secondary antibody solution [Goat-anti-Rabbit-594 (Thermo Fisher Scientific, A-11012) at 1:400 and Goat-anti-Mouse-488 (Thermo Fisher Scientific, A-11029) at 1:400] for 01:00:00 at 25 °C protected from light.
1h
Stain samples with Hoechst 33342 1.25 µg/mL in DBPS for 00:10:00 at 25 °C protected from light.
10m
Wash samples three times with DPBS, then mount coverslips on slides with ProLong Glass Antifade Mountant and seal with clear nail polish.
Imaging
Imaging
2h
2h
Image samples on a confocal microscope at 100x magnification with an oil objective.
Data analysis
Data analysis
Calculate Mander’s correlation coefficients with JACoP plugin in Fiji to assess the colocalization of signals from two channels.