TITLE:  LRRK2 and LAMP1 immunofluorescence staining in various cell lines
AUTHORS: Siyu Chen, eva karasmanis

[DESCRIPTION]
This protocol is being used to test the antibody   , as well as  . Please note that after multiple rounds of optimization, anti-LRRK2 antibody generates significant non-specific signals in the LRRK2 KO cell lines so this antibody may not be reliable for Immunofluorescence staining experiments. The protocol can be used for general IF experiments.


[BEFORE_START]
Important note:
While LAMP1 immunofluorescence staining robustly gives expected results, after multiple rounds of optimization, anti-LRRK2 antibody generates significant non-specific signals in the LRRK2 KO cell lines in our hand. This is a warning that this antibody may not be reliable for Immunofluorescence staining experiments. However, this protocol can be used widely for IF experiments.
Please check This google sheet to learn more about the cell lines and conditions tested for the antibody

[GUIDELINES]
GDB buffer was used as blocking buffer in the optimized protocol.
This protocol uses ibidi 8 Well Chamber µ-Slides and will coat them with Fibronectin. Standard volume would be 300 uL for each well. Volumes will need altering for wells, other plates and slides. 
All steps are performed at room temperature (RT) on the lab bench, except for methanol permeabilization.

[STEPS]
SECTION: Day 0: : Seed cells
1. Add 300 µL  of 11 ug/mL fibronectin into each ibidi well. Incubate at RT for 60 min.

SECTION: Day 0: : Seed cells
2. Rinse the wells with PBS for 3 times
SECTION: Day 0: : Seed cells
3. Make GDB buffer if necessary
 

SECTION: Day 0: : Seed cells
4. Seed adherent cells to 40-80% confluency in each well. Incubate at 37 °C   to get optimal seeding.
 

SECTION: Day 1: Drug treatment
5. Apply any drug treatments or controls and note time of additions before proceeding with fixing. As an example,    and    can be added at desired concentrations for 180 min 
 
In each well, add 1 in 1000 ( 1 millimolar (mM) for LLOME and 0.1 millimolar (mM) for CQ)
SECTION: Staining
6. Put 100% ethanol on ice before proceeding with next steps.
SECTION: Staining
7. Bring GDB buffer to Room temperature . Prepare and prewarm fixation buffer (4% sucrose, 3% PFA in 1xPBS) at 37 °C
 

SECTION: Staining
8. Get cells, aspirate media and immediately add prewarmed fixation buffer. Incubate for 10 min at 37 °C 
SECTION: Staining
9. Aspirate PFA, rinse 2x with PBS and wash two times with PBS for 5 minutes each, 10 min in total

SECTION: Staining
10. Quenching: Only necessary when staining the day of fixation. Incubate 3 times of 10 minutes (30 min in total) using 0.4%   (75 millimolar (mM))
SECTION: Staining
11. Optional step: For half of the samples, choose to add another permeablisation step with 300 µL 100% ethanol at -20°C for 20 min. Leave the rest at Room temperature in GDB buffer.
 

 

SECTION: Staining
12. Apsirate ethanol, wash 2x with PBS.
SECTION: Staining
13. Prepare GDB + 0.05%   freshly. Add 300 µL for 10 min.
5 µL in 10 mL GDB buffer. Can be stored at 4 °C for a couple of days.
SECTION: Staining
14. Aspirate Triton X-100, wash 2x with GDB.
SECTION: Staining
15. Block cells with 300 µL GDB for 30 min at Room temperature 

SECTION: Staining
16. During blocking, prepare final concentration of 1 ug/mL for both  and   solution with GDB and 50000 rpm, 10 min, 4 °C - each ibidi dish well requires 150 µL
 

SECTION: Staining
17. Take most of the supernatant and place in new tube. Mix to get even concentration (due to   in there, there will be a small clear precipitate)
SECTION: Staining
18. Aspirate blocking solution and incubate cells with 150 µL of primary antibody solution   at 4 °C on a table-top shaker

 

 

SECTION: Day 2
19. Bring GDB to Room temperature. Aspirate antibody solution and rinse 2x with GDB.
SECTION: Day 2
20. Wash 3x 5min Room temperature RT with GDB, 15 min in total

SECTION: Day 2
21. Prepare 1:500 Alexa-flour conjugated secondary antibody solution with GDB - each ibidi well requires 200 µL.
When LRRK2 and LAMP1 are co-stained,   and   are used at final concentration of 4 ug/mL. Lower concentration did not help with the issue of non-specific LRRK2 signal.

SECTION: Day 2
22. Incubate cells with 200 µL of secondary antibody solution for 90 min at Room temperature and protect from light with an ice box.
SECTION: Day 2
23. 15 minutes before the incubation is finished, add   to a final concentration of 1 ug/mL  and incubate until the last step finishes.

SECTION: Day 2
24. Rinse cells with 1xPBS for 5 times
SECTION: Day 2
25. Apply 2-4 drops of   hard mounting media in each well and swirl to make sure the bottom is fully covered.
SECTION: Day 2
26. Allow to air-dry for 10 min at Room temperature.
SECTION: Day 2
27. Image within 48 h of mounting or the sample will begin to deteriorate (bright debris impeding imaging) and visibly autofluoresce in red.