LRRK2 and LAMP1 immunofluorescence staining in various cell lines
Siyu Chen, eva karasmanis
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Abstract
This protocol is being used to test the antibody
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
Steps
Day 0: : Seed cells
Add 300µL of 11ug/mL fibronectin into each ibidi well. Incubate at RT for 1h 0m 0s.
Rinse the wells with PBS for 3 times
Make GDB buffer if necessary
| A | B | C | D | E |
|---|---|---|---|---|
| (0.1M) NaPi pH 7.4 | 15 | mL | 30 | mM |
| (5M) NaCl | 0.0045 | mL | 0.45 | mM |
| Gelatin | 0.1 | g | 0.2 | % |
| H2O | 34.9955 | mL | ||
| Total | 50 | mL |
Recipe to make GDB buffer
Seed adherent cells to 40-80% confluency in each well. Incubate at 37°C to get optimal seeding.
Day 1: Drug treatment
Apply any drug treatments or controls and note time of additions before proceeding with fixing. As an example, 3h 0m 0s
| A | B | C | D | E |
|---|---|---|---|---|
| LLOME | 339.27 | 1000 | 339.27 | mg/1mL |
| CQ | 515.86 | 100 | 51.586 | mg/1mL |
Drug stock recipe and concentrations
In each well, add 1 in 1000 ( 1millimolar (mM) for LLOME and 0.1millimolar (mM) for CQ)
Staining
Put 100% ethanol on ice before proceeding with next steps.
Bring GDB buffer to Room temperature . Prepare and prewarm fixation buffer (4% sucrose, 3% PFA in 1xPBS) at 37°C
Get cells, aspirate media and immediately add prewarmed fixation buffer. Incubate for 0h 10m 0s at 37°C
Aspirate PFA, rinse 2x with PBS and wash two times with PBS for 5 minutes each, 0h 10m 0s in total
Quenching: Only necessary when staining the day of fixation. Incubate 3 times of 10 minutes (0h 30m 0s in total) using 0.4% 75millimolar (mM))
Optional step: For half of the samples, choose to add another permeablisation step with 300µL 100% ethanol at -20°C for 0h 20m 0s. Leave the rest at Room temperature in GDB buffer.
Apsirate ethanol, wash 2x with PBS.
Prepare GDB + 0.05% 300µL for 0h 10m 0s.
5µL in 10mL GDB buffer. Can be stored at 4°C for a couple of days.
Aspirate Triton X-100, wash 2x with GDB.
Block cells with 300µL GDB for 0h 30m 0s at Room temperature
During blocking, prepare final concentration of 1ug/mL for both50000rpm,4°C - each ibidi dish well requires 150µL
Take most of the supernatant and place in new tube. Mix to get even concentration (due to
Aspirate blocking solution and incubate cells with 150µL of primary antibody solution at 4°C on a table-top shaker
Day 2
Bring GDB to Room temperature. Aspirate antibody solution and rinse 2x with GDB.
Wash 3x 5min Room temperature RT with GDB, 0h 15m 0s in total
Prepare 1:500 Alexa-flour conjugated secondary antibody solution with GDB - each ibidi well requires 200µL.
When LRRK2 and LAMP1 are co-stained, 4ug/mL. Lower concentration did not help with the issue of non-specific LRRK2 signal.
Incubate cells with 200µL of secondary antibody solution for 1h 30m 0s at Room temperature and protect from light with an ice box.
15 minutes before the incubation is finished, add 1ug/mL and incubate until the last step finishes.
Rinse cells with 1xPBS for 5 times
Apply 2-4 drops of
Allow to air-dry for 0h 10m 0s at Room temperature.
Image within 48 h of mounting or the sample will begin to deteriorate (bright debris impeding imaging) and visibly autofluoresce in red.
