TITLE: Immunofluorescence for detection of ALFA-tag in S. rosetta 
AUTHORS: Fredrick leon, David Booth

[DESCRIPTION]
Immunofluorescence (IF) allows for the visualization of protein localization in fixed-cell samples. This protocol builds upon commonly used IF methods, with a focus on retaining and minimally damaging S. rosetta's collar and flagella. It also introduces a new permeabilization buffer composed of LICOR Intercept blocking buffer for greater anti-ALFA tag nanobody specificity and signal. This protocol has been optimized for IF with the ALFA tag, but works well for common antibodies, such as DM1A and phalloidan.  This protocol was developed with essential input from Flora Rutaganira and Alain Garcia de las Bayones

[STEPS]
SECTION: Adsorb cells to Glass-Bottom Dish
5. Pipette 50 µL of 10 mg/mL onto the glass bottom of a glass-bottomed 96 well plate and incubate for 15 min at Room temperature.

  
  

SECTION: Adsorb cells to Glass-Bottom Dish
6. Remove the excess liquid and add 50 µL. Repeat twice more for a total of 3 washes.

  

SECTION: Adsorb cells to Glass-Bottom Dish
7. Gently pipette 50 µLonto the surface of the glass. Use a wide-bore pipette tip (or cut a 1000 µl pipette tip) to decrease shear forces on cells. Let the cells settle on the surface for 15 min.

SECTION: Cell Fixation
14. Gently apply 50 µL and incubate for 5 min 
 

SECTION: Cell Fixation
15. Gently apply 50 µLand incubate for 5 min 
SECTION: Cell Fixation
16. Gently apply 50 µL and proceed immediately to permeabilization
SECTION: Permeabilization and Blocking
17. Gently apply 50 µL and incubate for 15 min 


SECTION: Apply Stains
20. Gently apply 50 µL. Repeat one more for a total of 2 applications to ensure the antibodies are not too dilute. Incubate for 60 min 
 


SECTION: Apply Stains
21. Gently apply 50 µL. Repeat one more for a total of 2 applications to properly wash the cells of unbound antibodies. 
SECTION: Image Samples
22. With the cells in PEM buffer, proceed to imaging. 
SECTION: Grow cells
1. Seed cells at 10,000 cells/mL 24 hours hours in advance and grow at 22 °C  

SECTION: Grow cells
2. On the day of imaging, gently concentrate cells by centrifugation at 500 x g, 5 min, 22 °C 

SECTION: Grow cells
3. Carefully remove supernatant with a pipette, leaving the last 50-100 µL 
 


SECTION: Grow cells
4. Gently swirl the remaining supernatant and cells to resuspend the pellet. Do NOT vortex.
SECTION: Prepare buffers
8. Assemble The following buffers, and use all at room temperate

PFA fix buffer: Fixes the cells initially
 

SECTION: Prepare buffers
12. PermB buffer: Permeabilization buffer without methanol for washing cells and diluting antibodies
 
  
SECTION: Prepare buffers
11. PermB-meOH buffer: Permeabilizes the cells with a higher Tween concentration and methanol
 
   

SECTION: Prepare buffers
10. Gycline wash buffer : Quenches the remaining PFA from the previous fixation steps
 
 
SECTION: Prepare buffers
9. PFA/Tween fix buffer: Continues the fixation process and the Tween addition very gently permeabilizes cells             
 

SECTION: Prepare buffers
13. PEM buffer : For washing cells of excess antibodies and matching the reflective index of water immersion objectives
 
 
SECTION: Permeabilization and Blocking
19. Gently apply 50 µL
 

SECTION: Permeabilization and Blocking
18. During the incubation, dilute antibodies in PermB buffer