Effective and efficient cytoskeleton (actin and microtubules) fluorescence staining of adherent eukaryotic cells
Alfredo Leonardo Porfirio-Sousa, Tristan Henderson, Matthew W. Brown
immunofluorescence
protist
microscopy
confocal
tubulin
microtubules
actin
nucleus
Phalloidin
antibody
Hoechst
DNA
Abstract
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.
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 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 0h 15m 0s to under normal incubation conditions. Check on the inverted microscope to see if your cells have adhered.
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 0h 15m 0s to under normal incubation conditions. Check on the inverted microscope to see if your cells have adhered
Ensure Paraformaldehyde (8%) is at -80Room temperature.
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.
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. Add 500µL 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.
Fix cells by gently adding 500µL 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.
Incubate at Room temperature for 0h 10m 0s .
Gently aspirate liquid with a 1mL pipette and discard.
Rinse gently by adding 500µL PBS to chamber's side and allowing the liquid to gently flow down onto glass surface. Wash a total of three times for 0h 3m 0s each.
Gently aspirate liquid with a 1mL pipette and discard. Permeabilize cells by adding 500µL of 0.5% Triton™ X-100 and incubate for 0h 5m 0s at Room temperature.
Rinse gently by adding 500µL PBS to chamber's side and allowing the liquid to gently flow down onto glass surface. Wash a total of three times for 0h 3m 0s each.
Gently aspirate liquid with a 1mL pipette and discard. Add 500µL of Serum Blocking Buffer per chamber (this is the blocking agent) and incubate for 0h 10m 0s at 22Room temperature.
If Serum Blocking Buffer is not available, you may use 1X BSA Blocking Buffer as above.
Add 500µL of 1:500 primary antibody [monoclonal Anti-α-Tubulin antibody produced in mouse clone B-5-1-2] to the chamber slide and incubate 0h 30m 0s at 22Room temperature. This will bring your entire volume up to 1000µL.
For a negative control, For a negative control , add 500µL of PBS to the other chamber slide and incubate 0h 30m 0s at 22Room temperature. This will bring your entire volume up to 1000µL.
Add 2 drops of ActinGreen 488nm ReadyProbes Reagent (Thermo Fisher Scientific | R37110) to each chamber slide and incubate 0h 30m 0s at 22Room temperature.
Gently aspirate liquid with a 1mL pipette and discard.
Rinse gently by adding 500µL PBS to chamber's side and allowing the liquid to gently flow down onto glass surface. Wash a total of four times for0h 5m 0seach. Aspirate liquid completely after final wash.
Add 500µL of 1:1000 secondary antibody [Goat anti-Mouse IgG (H L) Secondary Antibody, Alexa 594] to each chamber and incubate for a 0h 15m 0s at 22Room temperature. Keep dark by covering with a box.
Add 2 drops of ActinGreen 488nm ReadyProbes Reagent (Thermo Fisher Scientific | R37110) to each chamber slide and incubate 0h 10m 0s at 22Room temperature. Keep dark by covering with a box.
Add 2 drops of NucBlue ReadyProbes (Thermo Fisher Scientific | R37605) to each chamber. Continue to incubate at 22Room temperature for 0h 10m 0s.
Rinse gently by adding 500µL PBS to chamber's side and allowing the liquid to gently flow down onto glass surface. Wash a total of three times for0h 5m 0seach. Aspirate liquid completely after final wash.
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 Room temperature for 0h 15m 0s. Keep dark by covering with a box.
Seal the edges of the cover slip with transparent nail lacquer. Let the nail lacquer dry for0h 15m 0s at Room temperature. Keep dark by covering with a box.
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.
