Aug 21, 2024

Immunofluorescence of GM2

 Forked from Immunofluorescence of RAB5 and FLAG-EEA1 puncta after Dynamin-1 and -2 inhibition with Dyngo4a
DOI
https://dx.doi.org/10.17504/protocols.io.36wgqj4d5vk5/v1
  • 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;
  • 4Harvard Medical School;
  • 5harvard university
Harper JW
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DOI: https://dx.doi.org/10.17504/protocols.io.36wgqj4d5vk5/v1
Protocol Citation: Hankum Park, Frances V Hundley, Harper JW 2024. Immunofluorescence of GM2. protocols.io https://dx.doi.org/10.17504/protocols.io.36wgqj4d5vk5/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: October 08, 2022
Last Modified: August 21, 2024
Protocol  Integer ID: 71042
Keywords: endosome, immunoflourescence, EEA1, RAB5, ASAPCRN, snapshots of early endosome, early endosome, purified endosome, immunofluorescence of gm2 selective purification, protein eea1, associated protein eea1, immunofluorescence protocol, immunofluorescence, gm2 selective purification, inhibitor dyngo4a, termed endo, protein
Funders Acknowledgements:
ASAP
Grant ID: 000282
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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
ABC
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
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
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
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
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
2h
Image samples on a confocal microscope at 100x magnification with an oil objective.
Data analysis
Calculate Mander’s correlation coefficients with JACoP plugin in Fiji to assess the colocalization of signals from two channels.