Aug 05, 2023

Public workspace LRRK2 and LAMP1 immunofluorescence staining in various cell lines

DOI
dx.doi.org/10.17504/protocols.io.bp2l6x991lqe/v1
  • 1UC San Diego, HHMI
Siyu Chen
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DOI: dx.doi.org/10.17504/protocols.io.bp2l6x991lqe/v1
Protocol Citation: Siyu Chen, eva karasmanis 2023. LRRK2 and LAMP1 immunofluorescence staining in various cell lines. protocols.io https://dx.doi.org/10.17504/protocols.io.bp2l6x991lqe/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: Other
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.
Created: August 03, 2023
Last Modified: May 31, 2024
Protocol Integer ID: 85948
Keywords: IF, LRRK2, RAW264.7, LAMP1, ASAPCRN
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Abstract
This protocol is being used to test the antibody ReagentRecombinant Anti-LRRK2 antibody [MJFF2 (c41-2)] (ab133474)Abcam , as well as ReagentAnti-LAMP1 antibody [1D4B] (ab25245)Abcam . 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.

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.
Materials
  • (0.1M) NaPi pH 7.4 3.1 g of NaH2PO4•H2O 0.9 g of Na2HPO4 (anhydrous) distilled H2O to make a volume of 1 L The pH of the final solution will be 7.4. This buffer can be stored for up to 1 mo at 4°C
  • GDB buffer 30mM NaPi (sodium phosphate) pH 7.4 0.45mM NaCl 0.2% porcine (or fish) gelatin In ddH2O
  • Kim wipes
  • Ethanol (100%, stored in dark chemicals cupboard)
  • Water (double-deionised H2O from Milli-Q, “MQ-H2O”)
Protocol materials
ReagentTriton X-100 Merck MilliporeSigma (Sigma-Aldrich)Catalog #X100
Step 13
ReagentGelatinMerck MilliporeSigma (Sigma-Aldrich)Catalog #G2500
Step 17
ReagentGoat Anti-Rat IgG H&L (Alexa Fluor® 488) (ab150157)Abcam
Step 21
ReagentFluorSave™ ReagentMerck MilliporeSigma (Sigma-Aldrich)Catalog #345789
Step 25
ReagentRecombinant Anti-LRRK2 antibody [MJFF2 (c41-2)] (ab133474)Abcam
Abstract, Step 16
ReagentAnti-LAMP1 antibody [1D4B] (ab25245)Abcam
Abstract, Step 16
ReagentChloroquine diphosphate saltMerck MilliporeSigma (Sigma-Aldrich)Catalog #C6628-25G
Step 5
ReagentLeu-Leu methyl ester hydrobromideMerck MilliporeSigma (Sigma-Aldrich)Catalog #L7393-500MG
Step 5
ReagentAmmonium chlorideMerck MilliporeSigma (Sigma-Aldrich)Catalog #254134
Step 10
ReagentGoat Anti-Rabbit IgG H&L (Alexa Fluor® 647) (ab150079)Abcam
Step 21
ReagentDAPI Thermo Fisher ScientificCatalog #D1306
Step 23
Safety warnings
N/A
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
Day 0: : Seed cells
Day 0: : Seed cells
1h
1h
Add Amount300 µL of Concentration11 ug/mL fibronectin into each ibidi well. Incubate at RT for Duration01:00:00 .

1h
Rinse the wells with PBS for 3 times
Make GDB buffer if necessary
StockAmount neededCFinal conc.E
(0.1M) NaPi pH 7.415mL30mM
(5M) NaCl0.0045mL0.45mM
Gelatin0.1g0.2%
H2O34.9955mL
Total50mL
Recipe to make GDB buffer

Seed adherent cells to 40-80% confluency in each well. Incubate at Temperature37 °C DurationOvernight to get optimal seeding.
Note
For RAW264.7 cells, 6x10^4 in 300uL is a good starting point. Less would be needed for other typical cell lines since macrophage cells are smaller.
It is suggested to start with two different cell concentrations for the first time. Please refer to this page for more information

Day 1: Drug treatment
Day 1: Drug treatment
3h
3h
Apply any drug treatments or controls and note time of additions before proceeding with fixing. As an example, ReagentChloroquine diphosphate saltMerck MilliporeSigma (Sigma-Aldrich)Catalog #C6628-25G and ReagentLeu-Leu methyl ester hydrobromideMerck MilliporeSigma (Sigma-Aldrich)Catalog #L7393-500MG can be added at desired concentrations for Duration03:00:00
DrugsMW - g/molmMweight in 1 mLE
LLOME339.271000339.27mg/1mL
CQ515.8610051.586mg/1mL
Drug stock recipe and concentrations
In each well, add 1 in 1000 ( Concentration1 millimolar (mM) for LLOME and Concentration0.1 millimolar (mM) for CQ)
3h
Staining
Staining
25m
25m
Put 100% ethanol on ice before proceeding with next steps.
Bring GDB buffer to TemperatureRoom temperature . Prepare and prewarm fixation buffer (4% sucrose, 3% PFA in 1xPBS) at Temperature37 °C
Note
3% PFA is preferred from 4% as it reduces autofluorescence
Need Amount375 µL ReagentPierce™ 16% Formaldehyde (w/v) Methanol-freeThermo Fisher ScientificCatalog #28906 and Amount0.08 g ReagentSucroseMerck MilliporeSigma (Sigma-Aldrich)Catalog #S0389 , dissove with Amount2 mL PBS.

Get cells, aspirate media and immediately add prewarmed fixation buffer. Incubate for Duration00:10:00 at Temperature37 °C
10m
Aspirate PFA, rinse 2x with PBS and wash two times with PBS for 5 minutes each, Duration00:10:00 in total

10m
Quenching: Only necessary when staining the day of fixation. Incubate 3 times of 10 minutes (Duration00:30:00 in total) using 0.4% ReagentAmmonium chlorideMerck MilliporeSigma (Sigma-Aldrich)Catalog #254134 (Concentration75 millimolar (mM) )
30m
Optional step: For half of the samples, choose to add another permeablisation step with Amount300 µL 100% ethanol at -20°C for Duration00:20:00 . Leave the rest at TemperatureRoom temperature in GDB buffer.
Note
This step helps to increase contrast when imaging ReagentRecombinant Anti-LRRK2 antibody [MJFF2 (c41-2)] (ab133474)Abcam , but doesn't help with the issue of non-specific signals when LRRK2 KO cell lines are used.

Note
ethanol/methanol permeablisation at Temperature-20 °C will disrupt the microtubule staining. Apply this step with caution when other antibodies are used.


20m
Apsirate ethanol, wash 2x with PBS.
Prepare GDB + 0.05% ReagentTriton X-100 Merck MilliporeSigma (Sigma-Aldrich)Catalog #X100 freshly. Add Amount300 µL for Duration00:10:00 .
Amount5 µL in Amount10 mL GDB buffer. Can be stored at Temperature4 °C for a couple of days.
10m
Aspirate Triton X-100, wash 2x with GDB.
Block cells with Amount300 µL GDB for Duration00:30:00 at TemperatureRoom temperature

30m
During blocking, prepare final concentration of Concentration1 ug/mL for bothReagentRecombinant Anti-LRRK2 antibody [MJFF2 (c41-2)] (ab133474)Abcam and ReagentAnti-LAMP1 antibody [1D4B] (ab25245)Abcam solution with GDB and Centrifigation50000 rpm, 4°C, 00:10:00 - each ibidi dish well requires Amount150 µL
Note
Amount150 µL is the minimum required amount. Amount200 µL would be sufficient and optimal to cover the entire well

10m
Take most of the supernatant and place in new tube. Mix to get even concentration (due to ReagentGelatinMerck MilliporeSigma (Sigma-Aldrich)Catalog #G2500 in there, there will be a small clear precipitate)
Aspirate blocking solution and incubate cells with Amount150 µL of primary antibody solution DurationOvernight at Temperature4 °C on a table-top shaker

Note
Primary antibody incubation can be as short as 2 hours without affecting the final outcome

Note
When incubating overnight, consider wrapping up the dish with parafilm and/or put the dish in a humidity chamber to prevent the well from drying up

Day 2
Day 2
15m
15m
Bring GDB to TemperatureRoom temperature . Aspirate antibody solution and rinse 2x with GDB.
Wash 3x 5min TemperatureRoom temperature RT with GDB, Duration00:15:00 in total

15m
Prepare 1:500 Alexa-flour conjugated secondary antibody solution with GDB - each ibidi well requires Amount200 µL .
When LRRK2 and LAMP1 are co-stained, ReagentGoat Anti-Rabbit IgG H&L (Alexa Fluor® 647) (ab150079)Abcam and ReagentGoat Anti-Rat IgG H&L (Alexa Fluor® 488) (ab150157)Abcam are used at final concentration of Concentration4 ug/mL . Lower concentration did not help with the issue of non-specific LRRK2 signal.

Incubate cells with Amount200 µL of secondary antibody solution for Duration01:30:00 at TemperatureRoom temperature and protect from light with an ice box.
1h 30m
15 minutes before the incubation is finished, add ReagentDAPI Thermo Fisher ScientificCatalog #D1306 to a final concentration of Concentration1 ug/mL and incubate until the last step finishes.

Rinse cells with 1xPBS for 5 times
Apply 2-4 drops of ReagentFluorSave™ ReagentMerck MilliporeSigma (Sigma-Aldrich)Catalog #345789 hard mounting media in each well and swirl to make sure the bottom is fully covered.
Allow to air-dry for Duration00:10:00 at TemperatureRoom temperature .
10m
Image within 48 h of mounting or the sample will begin to deteriorate (bright debris impeding imaging) and visibly autofluoresce in red.