TITLE: Effective and efficient cytoskeleton (actin and microtubules) fluorescence staining of adherent eukaryotic cells
AUTHORS: Matthew Brown

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">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. </div><br/><br/><div class = "text-block">Protocol is adapted from the following citations. </div><br/><br/><br/><br/></div>

[STEPS]
?. 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.
?. 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  to   under normal incubation conditions. Check on the inverted microscope to see if your cells have adhered
?. Ensure methanol at  for at least  to so that it is at   .
-80 °C
-80 °C
?. 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  to  under normal incubation conditions. Check on the inverted microscope to see if your cells have adhered.
?. If cells were grown on agar, remove agar block gently. Be sure to remove all agar chunks.
?. Aspirate liquid VERY gently and discard to bleach solution. Cells should still be attached to the chamber slide.
?. Fix cells in  Methanol (100%), by gently adding  to chamber's side and allowing the liquid to gently flow down onto glass surface.
-80 °C
1 mL
?. Gently aspirate liquid with a 1mL pipette and discard.
?. Incubate at room temperature for 2 minutes.
22 Room temperature
?. Rinse gently by adding  PBS to chamber's side and allowing the liquid to gently flow down onto glass surface.  Wash a total of three times foreach.
500 µl
?. Add  of Serum Blocking Buffer per chamber (this is the blocking agent) and incubate for  at .
500 µl
22 Room temperature
?. If  Serum Blocking Buffer  is not available, you may use 1X BSA Blocking Buffer as above.
?. Add  of  1:500 primary antibody [monoclonal Anti-α-Tubulin antibody produced in mouse clone B-5-1-2] to the chamber slide and incubate  at . This will bring your entire volume up to 1000µL.
500 µl
22 Room temperature
?. Add 2 drops of ActinGreen 488nm ReadyProbes Reagent (Thermo Fisher Scientific | R37110) to each chamber slide and incubate  at .
22 Room temperature
?. For a negative control, add   of PBS to the other chamber slide and incubate  at . This will bring your entire volume up to 1000µL.
500 µl
22 Room temperature
?. Rinse gently by adding  PBS to chamber's side and allowing the liquid to gently flow down onto glass surface.  Wash a total of four times foreach. Aspirate liquid completely after final wash.
500 µl
?. Gently aspirate liquid with a 1mL pipette and discard.
?. Add  of 1:1000 secondary antibody [Goat anti-Mouse IgG (H L) Secondary Antibody, Alexa 594] to each chamber and incubate for a  at . Keep dark by covering with a box.
500 µl
22 Room temperature
?. Add 2 drops of ActinGreen 488nm ReadyProbes Reagent (Thermo Fisher Scientific | R37110) to each chamber slide and incubate  at .  Keep dark by covering with a box.
22 Room temperature
?. Add 2 drops of NucBlue ReadyProbes (Thermo Fisher Scientific | R37605) to each chamber. Continue to incubate at  for .
22 Room temperature
?. Rinse gently by adding  PBS to chamber's side and allowing the liquid to gently flow down onto glass surface.  Wash a total of three times foreach. Aspirate liquid completely after final wash.
500 µl
?. Remove culture slide chamber sides with removal tool included with Lab-Tek™ II Chamber Slide (Thermo Fisher Scientific - 154461) kit.
?. 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  for . Keep dark by covering with a box.
0 Room temperature
?. Seal the edges of the cover slip with transparent nail lacquer. Let the nail lacquer dry for at  . Keep dark by covering with a box.
0 Room temperature
?. 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 in  in the dark.
4 °C
?. 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.