TITLE: Immunofluorescence of RAB5 and FLAG-EEA1 puncta after Dynamin-1 and -2 inhibition with Dyngo4a
AUTHORS: Hankum Park, Frances V Hundley, Harper     JW

[DESCRIPTION]
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.



[STEPS]
SECTION: Preparation of coverslips
1. Coat No.1.5 coverslips in 0.01% poly-L-lysine solution.  Incubate at 37 °C  for 15 min 

SECTION: Preparation of coverslips
2. Aspirate poly-L-lysine solution and wash coverslips three times with sterile DPBS.
SECTION: Preparation of coverslips
3. Dry coverslips at 37 °C for 15 min  .

SECTION: Seed cells
4. 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.  
SECTION: Dyngo4a treatment
5. The next day, check that cells are approximately 70% confluent.  
SECTION: Dyngo4a treatment
6. Aliquot and warm serum-free DMEM to 37 °C . 

SECTION: Dyngo4a treatment
7. 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%.
 

 


SECTION: Dyngo4a treatment
8. Aspirate existing media from cells growing on coverslips, and add new media containing either DMSO or Dyngo4a.  Return cells to incubator for 180 min 

SECTION: Dyngo4a treatment
9. 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.
SECTION: Sample fixation and staining
10. Fix cells in 4% paraformaldehyde solution in DPBS for 15 min at 25 °C .

SECTION: Sample fixation and staining
11. Wash samples three times in DPBS.  Block samples for 60 min at 25 °C in blocking buffer (1% BSA, 0.15% Triton X-100, DPBS).

SECTION: Sample fixation and staining
12. 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]   at 4 °C .  Include single primary antibody controls and no primary antibody controls.

SECTION: Sample fixation and staining
13. 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 60 min at 25 °C  protected from light. 

SECTION: Sample fixation and staining
14. Stain samples with Hoechst 33342 1.25 µg/mL in DBPS for 10 min at 25 °C  protected from light.  

SECTION: Sample fixation and staining
15. Wash samples three times with DPBS, then mount coverslips on slides with ProLong Glass Antifade Mountant and seal with clear nail polish.
SECTION: Imaging
16. Image samples on a confocal microscope at 100x magnification with an oil objective.
SECTION: Data analysis
17. Calculate Mander’s correlation coefficients with JACoP plugin in Fiji to assess the colocalization of signals from two channels.