TITLE: Immunofluorescence imaging of cells expressing GolgiTAG (TMEM115-3HA) or HA-tagged non-Golgi annotated proteins
AUTHORS: Rotimi 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 HA-tagged Golgi proteins (like TMEM115-3HA, or GolgiTAG), by assessing their colocalisation with known Golgi markers such as GM130, GOLGIN97 and ACBD3. Furthermore, this method can be used to investigate whether HA-tagged, non-Golgi annotated proteins transiently expressed in cells localise at the Golgi, by assessing their colocalisation with ACBD3.

[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: Transient transfection of cells for immunofluorescence microscopy
5. For each plasmid, prepare a transfection mix in a sterile 1.5 mL Eppendorf tube containing:
2 µg plasmid (see Table 1 for list of plasmids).
6 µL 1 mg/mLPEI Max 40K (dissolved in distilled water and filter sterile).
300 µL OptiMem.
SECTION: Transient transfection of cells for immunofluorescence microscopy
6. Incubate the transfection mix at Room temperature for 30 min.
SECTION: Transient transfection of cells for immunofluorescence microscopy
7. Add the transfection mix dropwise to the cells from step 3 using a P1000 sterile pipette.
SECTION: Transient transfection of cells for immunofluorescence microscopy
8. Incubate cells at 37 °C for 1440 min.
SECTION: Preparing cells for Immunofluorescence imaging
9. Remove media completely using an aspirator and wash cells 3 times with 3 mL PBS added wit 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
10. Fix cells by adding 4% (w/v) PFA in PBS.

SECTION: Preparing cells for Immunofluorescence imaging
11. Incubate at Room temperature for 10 min.
SECTION: Preparing cells for Immunofluorescence imaging
12. 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
13. Block with 1% (w/v) BSA in PBS at Room temperature for 30 min.
SECTION: Preparing cells for Immunofluorescence imaging
14. Prepare the primary antibody dilutions in 0.2% BSA (w/v) in PBS + 0.02% (w/v) sodium azide (See Table 2 for a list of antibodies and their working dilution).
 

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

SECTION: Preparing cells for Immunofluorescence imaging
16. 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
17. Prepare the secondary antibody dilutions in 0.2% BSA (w/v) in PBS + 0.02% (w/v) sodium azide (See Table 3 for a list of antibodies and their working dilution).
SECTION: Preparing cells for Immunofluorescence imaging
18. Incubate cells with diluted secondary antibodies (20 µL per coverslip) at Room temperature in the dark for 60 min.
 


SECTION: Preparing cells for Immunofluorescence imaging
19. Wash cells 3 times in 0.2% (w/v) BSA in PBS + 0.02% (w/v) sodium azide. (5 min per wash).
SECTION: Preparing cells for Immunofluorescence imaging
20. Rinse cells by dipping briefly in MilliQ water and gently dry on Kleenex wipes.
SECTION: Preparing cells for Immunofluorescence imaging
21. Add a drop of Gold anti-glare oil (with DAPI for nucleus staining) (Invitrogen #2086915) on a microscope glass slide labelled with the sample ID.
SECTION: Preparing cells for Immunofluorescence imaging
22. Mount cover slips from step 20 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 in the dark.
SECTION: Preparing cells for Immunofluorescence imaging
23. Slides can be stored at 4 °C  or viewed immediately on a confocal microscope.
  

SECTION: Preparing cells for Immunofluorescence imaging
9.1. Wash cells with 3 mL PBS added wit 0.2% (w/v) BSA and 0.02% (w/v) sodium azide for 5 min(1/3).
SECTION: Preparing cells for Immunofluorescence imaging
9.3. Wash cells with 3 mL PBS added wit 0.2% (w/v) BSA and 0.02% (w/v) sodium azide for 5 min(3/3).
SECTION: Preparing cells for Immunofluorescence imaging
9.2. Wash cells with 3 mL PBS added wit 0.2% (w/v) BSA and 0.02% (w/v) sodium azide for 5 min(2/3).
SECTION: Preparing cells for Immunofluorescence imaging
16.1. Wash the coverslips with 0.2% (w/v) BSA in PBS + 0.02% sodium azide for 5 min(1/3).
SECTION: Preparing cells for Immunofluorescence imaging
16.3. Wash the coverslips with 0.2% (w/v) BSA in PBS + 0.02% sodium azide for 5 min(3/3).
SECTION: Preparing cells for Immunofluorescence imaging
16.2. Wash the coverslips with 0.2% (w/v) BSA in PBS + 0.02% sodium azide for 5 min(2/3).
SECTION: Preparing cells for Immunofluorescence imaging
19.1. Wash cells  in 0.2% (w/v) BSA in PBS + 0.02% (w/v) sodium azide for 5 min(1/3).
SECTION: Preparing cells for Immunofluorescence imaging
19.2. Wash cells  in 0.2% (w/v) BSA in PBS + 0.02% (w/v) sodium azide for 5 min(2/3).
SECTION: Preparing cells for Immunofluorescence imaging
19.3. Wash cells  in 0.2% (w/v) BSA in PBS + 0.02% (w/v) sodium azide for 5 min(3/3).