May 09, 2024
Colocalisation imaging of endogenous TMEM192 with lysosomal and mitochondria markers
- Rotimi Y. Fasimoye1,2,
- Dario R. Alessi1,2
- 1Aligning Science Across Parkinson's;
- 2Medical Research Council Protein Phosphorylation and Ubiquitylation Unit, School of Life Sciences, University of Dundee, Dow Street, Dundee DD1 5EH, UK
Protocol Citation: Rotimi Y. Fasimoye, Dario R. Alessi 2024. Colocalisation imaging of endogenous TMEM192 with lysosomal and mitochondria markers. protocols.io https://dx.doi.org/10.17504/protocols.io.q26g71zykgwz/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: March 14, 2024
Last Modified: May 31, 2024
Protocol Integer ID: 96907
Keywords: ASAPCRN
Funders Acknowledgement:
Aligning Science Across Parkinson's
Grant ID: ASAP-000463
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Abstract
Immunofluorescent (IF) microscopy is a powerful tool used in cellular and molecular biology to monitor the subcellular localisation of proteins. By combining the advantages of immunostaining and confocal light microscope, IF microscopy can be used to assess the colocalization of two or more proteins within the cell. Here, we describe a method that can be used to verify the correct localisation of endogenously expressed TMEM192, by assessing their colocalization with LAMP1 (a lysosomal marker) and ATPB1 (a mitochondrial marker). Furthermore, our data showed that the anti-TMEM192 antibody is compatible for immunofluorescence assay.
Attachments
Materials
Materials:
1. Cell lines
- HEK293ATCCCatalog #CRL-1573 , RRID:CVCL_0045)
2.Antibodies
- See Tables 1 and 2
Table 1: List of primary antibodies
| A | B | C | D | |
| Antibody | Company | Cat. number | Host species | |
| TMEM192 | Abcam | Ab232600 | Rabbit | |
| LAMP1 | Santa Cruz | Sc-20011 | Mouse | |
| ATPB | Abcam | Ab14730 | Mouse |
Table 2: List of fluorophore-conjugated secondary antibodies
| A | B | C | D | E | |
| Antibody | Conjugated Fluorophore | Company | Cat. number | Host Species | |
| anti-Mouse | Alexa 488 | Invitrogen | A21206 | Donkey | |
| anti-Rabbit | Alexa 594 | Invitrogen | A11012 | Goat |
3. Media and Reagents
- Growth Media:
- DMEM (Gibco™ #11960-085)Gibco - Thermo FischerCatalog #11960085
- 10% (v/v) Fetal Bovine SerumMerck MilliporeSigma (Sigma-Aldrich)Catalog #F7524 Batch# BCBW6817)
- 1% L-Glutamine (200mM)Thermo Fisher ScientificCatalog #25030024
- 1% Penicillin-StreptomycinGibco - Thermo FisherCatalog #15140122
- DPBS no calcium no magnesiumGibco - Thermo FischerCatalog #14190169
- Bovine Serum Albumin Fraction VMerck MilliporeSigma (Sigma-Aldrich)Catalog #10735094001 Sodium azideMerck MilliporeSigma (Sigma-Aldrich)Catalog #S2002 Poly-L-lysineMerck MilliporeSigma (Sigma-Aldrich)Catalog #P4832 Hoechst 33342Thermo Fisher ScientificCatalog #62249
- VECTASHIELD antifading Mounting media (|Vector Laboratories, H1000)
4. Equipment
- Incubator with FPI-sensor system and display controller MB1 (BINDER GmbH. Model: CB150. Power Output: 1.40kW, 230V, 6.1 Amp)
- Leica TCS SP8 MP Multiphoton Microscope.
- See-saw rocker (VWR SSL4, or equivalent).
5. Consumables
- Nunc™ Cell-Culture Treated Multidishes, 6 wellThermo FisherCatalog #140675 .
- Cover glasses squareVWR InternationalCatalog #631-0125
- Microscope slides SuperFrost®VWR InternationalCatalog #631-0114 )
- Standard 1ml and 200µl Pipette tips (Greiner bio-one. Catalog# 686271 and 685261 respectively).
Seeding cells for immunofluorescence microscopy
Seeding cells for immunofluorescence microscopy
1h
Coat coverslips (sterilised in 100% ethanol prior to use) with poly-L-lysine by immersing the coverslips in poly-L-lysine solution for 01:00:00 .
1h
Rinse the coated coverslips in media and place in a 6-well plate (one coverslip in each well).
Seed cells to 50-60% confluency in growth media on coated coverslips from step 2.
Incubate Overnight .
Preparing cells for Immunofluorescence imaging
Preparing cells for Immunofluorescence imaging
3h 25m
Remove media completely using an aspirator and wash cells 3 times with 3 mL PBS added with 0.2% (w/v) BSA and 0.02% (w/v) sodium azide (00:05:00 per wash on a see-saw rocker).
5m
Fix cells by adding 4% (w/v) PFA in PBS and Incubate at Room temperature for 00:10:00 .
10m
Permeabilise cells with 1% (v/v) NP-40 in PBS + 0.2% (w/v) BSA + 0.02% (w/v) sodium azide.
Block with 3% (w/v) BSA in PBS at Room temperature for 00:30:00 .
30m
Prepare the primary antibody dilutions in 0.2% BSA (w/v) in PBS + 0.02% (w/v) sodium azide (See Table 1 for a list of antibodies and their working dilution).
| A | B | C | D | E | |
| Antibody | Company | Cat. number | Host Species | dilution | |
| TMEM192 | Abcam | Ab185545 | Rabbit | 1:1000 | |
| LAMP1 | Santa Cruz | Sc-20011 | Mouse | 1:1000 | |
| ATPB | Abcam | Ab14730 | Mouse | 1:1000 |
Table 1: List of primary antibodies
Note
Primary antibodies raised in different species are combined for co-staining, as follows:
- Mouse anti-LAMP1 and Rabbit anti-TMEM192
- Mouse anti-ATPB and Rabbit anti-TMEM192
Incubate cells at Room temperature with diluted primary antibodies for 01:00:00 .
Note
This should be done in a humid chamber to avoid samples drying out. Cover a glass plate with parafilm and add20 µL of primary antibody dilution to the relevant labelled area on the parafilm. Using tweezers, place each coverslip on the primary antibody solution (facing downward, so the cells are in contact with the antibody).
1h
Wash the coverslips 3 times with 0.2% (w/v) BSA in PBS + 0.02% sodium azide. (00:05:00 per wash).
5m
Prepare a combination of Secondary antibodies as described below (see Table 2 for more information about the secondary antibodies). Antibodies are diluted in PBS +0.2%BSA+0.02% sodium azide.
- anti-Mouse Alexa 488 (1:500) and anti-Rabbit Alexa 594 (1:500).
| A | B | C | D | E | |
| Antibody | Conjugated Fluorophore | Company | Cat. number | Host Species | |
| anti-Mouse | Alexa 488 | Invitrogen | A21206 | Donkey | |
| anti-Rabbit | Alexa 594 | Invitrogen | A11012 | Goat |
Table 2: List of fluorophore-conjugated secondary antibodies
Add 0.5 µL Hoechst 33342 solution for nuclear staining.
Incubate cells at Room temperature with diluted secondary antibodies for 01:00:00 . Do this in a humid chamber on a piece of Parafilm. Put a 60 µL drop of diluted antibodies on the parafilm. Carefully place coverslip on the droplet, with the side containing attached cells, facing inward, making contact with the droplet.
1h
Wash cells, 3 times, with 3 mL PBS +0.2%BSA+0.02% sodium azide.
Rinse cells by dipping briefly in MilliQ water and gently dry on Kleenex wipes.
Label microscope glass slides (preferably the one with frosted side) according to the primary antibody used. Take note of the emission wavelength of the probe on the secondary antibodies.
Add a drop of VECTASHIELD antifading Mounting media.
Mount cover slip (containing cells) on the glass slide, ensuring that the side containing the cells is facing inward, making contact with the oil. Allow to dry for 00:30:00 , ensuring slides are prevented from direct light.
30m
Store slides at 4 °C or view immediately on a confocal microscope.
Figure 1: Immunofluorescence images of HEK293 cells showing localisation of endogenous TMEM192 with LAMP1 (a lysosomal marker) and ATPB1 (a mitochondrial marker). Scale bar is 2 µm.