TITLE: Effective and efficient cytoskeleton (actin and microtubules) fluorescence staining of adherent eukaryotic cells
AUTHORS: Alfredo Leonardo Porfirio-Sousa, Tristan Henderson, Matthew Brown

[DESCRIPTION]
Eukaryotic microbes, protists, are highly diverse organisms with complex cytoskeletal elements used for movement consisting mostly of actin-myosin and microtubules. In order to visualize the cytoskeletal elements researchers may take a microscopical approach based on immunocytochemistry. Presented here is an efficient and effective for staining and visualizing actin microfilaments stained with phalloidin, nuclei stained with Hoechst 33342, and microtubules labeled using an alpha tubulin antibody. This protocol was developed for amoeboid protists, but will likely work on other adherent eukaryotic cells. 


Protocol is adapted from the following citations. 
 

 

 



[GUIDELINES]
Follow and adhere to all manufacturer's guidelines and warnings. Users must understand the MSDS data for each reagent before proceeding. 

If staining only for Actin and nucleus, and not for microtubules, skip steps 9, 10, 11, 12, and 16.

[STEPS]
2. Ensure Paraformaldehyde (8%) is at -80 Room temperature.

1. Move cells onto a chamber culture slide (Lab-Tek™ II Chamber Slide - Thermo Fisher Scientific - 154461) according to how the cells are being grown, see below.
1.1. If cells are growing on agar plates, cut block where there is dense area of cells. Place upside down on chamber culture slide (Lab-Tek™ II Chamber Slide - Thermo Fisher Scientific - 154461 or Nest Scientific 2 well slide 230122). Add 500µl of liquid media (same media as agar is made) and allow to sit for 15 min to   under normal incubation conditions. Check on the inverted microscope to see if your cells have adhered. 

1.2. If cells are growing in liquid media in a tissue culture flask, scrap cells with a cell scraper to dislodge cells from tissue culture flask. Move 1 mL to each chamber of the chamber culture slide (Lab-Tek™ II Chamber Slide - Thermo Fisher Scientific - 154461). Allow to sit for 15 min to    under normal incubation conditions. Check on the inverted microscope to see if your cells have adhered
3. Prepare all reagents as listed in the materials section. Prepare FRESH primary and secondary antibody dilutions before you proceed. The blocking buffer may be made in bulk beforehand. 
4. If cells were grown on agar, remove agar block gently. Be sure to remove all agar chunks. 
5. Aspirate liquid VERY gently and discard to bleach solution. Add 500 µL of liquid media (same media the cells were growing or same media as agar is made) to chamber's side and allowing the liquid to gently flow down onto glass surface. Cells should still be attached to the chamber slide. 

6. Fix cells by gently adding 500 µL of Paraformaldehyde (8%) at Room temperature to chamber's side and allowing the liquid to gently flow down onto glass surface. This will bring the solution to 4% Paraformaldehyde.

6.1. Incubate at Room temperature   for 10 min . 

7. Gently aspirate liquid with a 1mL pipette and discard. 
8. Rinse gently by adding 500 µL PBS to chamber's side and allowing the liquid to gently flow down onto glass surface.  Wash a total of three times for 3 min each. 

11. Gently aspirate liquid with a 1mL pipette and discard. Add 500 µL of Serum Blocking Buffer per chamber (this is the blocking agent) and incubate for 10 min at 22 Room temperature. 

11.1. If  Serum Blocking Buffer  is not available, you may use 1X BSA Blocking Buffer as above. 
12. Add 500 µL of  1:500 primary antibody [monoclonal Anti-α-Tubulin antibody produced in mouse clone B-5-1-2] to the chamber slide and incubate 30 min at 22 Room temperature. This will bring your entire volume up to 1000µL. 

12.1. For a negative control, add  500 µL of PBS to the other chamber slide and incubate 30 min at 22 Room temperature. This will bring your entire volume up to 1000µL. 

13. Add 2 drops of ActinGreen 488nm ReadyProbes Reagent (Thermo Fisher Scientific | R37110) to each chamber slide and incubate 30 min at 22 Room temperature. 
14. Gently aspirate liquid with a 1mL pipette and discard. 
15. Rinse gently by adding 500 µL PBS to chamber's side and allowing the liquid to gently flow down onto glass surface.  Wash a total of four times for5 mineach. Aspirate liquid completely after final wash. 
16. Add 500 µL of 1:1000 secondary antibody [Goat anti-Mouse IgG (H L) Secondary Antibody, Alexa 594] to each chamber and incubate for a 15 min at 22 Room temperature. Keep dark by covering with a box. 

17. Add 2 drops of ActinGreen 488nm ReadyProbes Reagent (Thermo Fisher Scientific | R37110) to each chamber slide and incubate 10 min at 22 Room temperature.  Keep dark by covering with a box. 
18. Add 2 drops of NucBlue ReadyProbes (Thermo Fisher Scientific | R37605) to each chamber. Continue to incubate at 22 Room temperature for 10 min. 

19. Rinse gently by adding 500 µL PBS to chamber's side and allowing the liquid to gently flow down onto glass surface.  Wash a total of three times for5 mineach. Aspirate liquid completely after final wash. 
20. Remove culture slide chamber sides with removal tool included with Lab-Tek™ II Chamber Slide (Thermo Fisher Scientific - 154461) kit. 
21. Mount your sample using a drop of Fluoromount-G (Thermo Fisher Scientific | 00-4958-02) (~100µL) and place a clean 1.5H  cover slip (22x22mm) on one side of the chamber area and allow the coverslip to gently set down to avoid air bubbles. Allow to incubate at Room temperature for 15 min. Keep dark by covering with a box. 

22. Seal the edges of the cover slip with transparent nail lacquer. Let the nail lacquer dry for15 min at  Room temperature. Keep dark by covering with a box. 

23. Visualize slide on a fluorescence microscope with DAPI, GFP, and TexasRed cubes or on a confocal microscope with 405, 488, 532/561 nm excitation lasers. Store slides horizontally in 4 °C in the dark.

9. Gently aspirate liquid with a 1mL pipette and discard. Permeabilize cells by adding 500 µL of 0.5% Triton™ X-100 and incubate for 5 min at Room temperature.   

10. Rinse gently by adding 500 µL PBS to chamber's side and allowing the liquid to gently flow down onto glass surface.  Wash a total of three times for 3 min each.