TITLE: Colocalisation imaging of endogenous TMEM192 with lysosomal and mitochondria markers
AUTHORS: Rotimi Y. Fasimoye, Dario R. Alessi

[DESCRIPTION]
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.

[STEPS]
SECTION: Seeding cells for immunofluorescence microscopy
1. Coat coverslips (sterilised in 100% ethanol prior to use) with poly-L-lysine by immersing the coverslips in poly-L-lysine solution for 60 min. 
SECTION: Seeding cells for immunofluorescence microscopy
2. Rinse the coated coverslips in media and place in a 6-well plate (one coverslip in each well).
SECTION: Seeding cells for immunofluorescence microscopy
3. Seed cells to 50-60% confluency in growth media on coated coverslips from step 2.
SECTION: Seeding cells for immunofluorescence microscopy
4. Incubate  .

SECTION: Preparing cells for Immunofluorescence imaging
5. 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 (5 min per wash on a see-saw rocker). 

SECTION: Preparing cells for Immunofluorescence imaging
6. Fix cells by adding 4% (w/v) PFA in PBS and Incubate at Room temperature for 10 min.

SECTION: Preparing cells for Immunofluorescence imaging
7. Permeabilise cells with 1% (v/v) NP-40 in PBS + 0.2% (w/v) BSA + 0.02% (w/v) sodium azide.
SECTION: Preparing cells for Immunofluorescence imaging
8. Block with 3% (w/v) BSA in PBS at Room temperature for 30 min.

SECTION: Preparing cells for Immunofluorescence imaging
9. 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).

 
 
 
 

SECTION: Preparing cells for Immunofluorescence imaging
10. Incubate cells at Room temperaturewith diluted primary antibodies for 60 min. 


 

SECTION: Preparing cells for Immunofluorescence imaging
11. Wash the coverslips 3 times with 0.2% (w/v) BSA in PBS + 0.02% sodium azide. (5 min per wash). 

SECTION: Preparing cells for Immunofluorescence imaging
12. 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). 

 
 
SECTION: Preparing cells for Immunofluorescence imaging
13. Add 0.5 µL Hoechst 33342 solution for nuclear staining.

SECTION: Preparing cells for Immunofluorescence imaging
14. Incubate cells at Room temperature with diluted secondary antibodies for 60 min. 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.

SECTION: Preparing cells for Immunofluorescence imaging
15. Wash cells, 3 times, with 3 mL PBS +0.2%BSA+0.02% sodium azide.

SECTION: Preparing cells for Immunofluorescence imaging
16. Rinse cells by dipping briefly in MilliQ water and gently dry on Kleenex wipes.
SECTION: Preparing cells for Immunofluorescence imaging
17. 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.
SECTION: Preparing cells for Immunofluorescence imaging
18. Add a drop of VECTASHIELD antifading Mounting media.
SECTION: Preparing cells for Immunofluorescence imaging
19. 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 30 min, ensuring slides are prevented from direct light.

SECTION: Preparing cells for Immunofluorescence imaging
20. Store slides at 4 °Cor view immediately on a confocal microscope.