Updates: Modified by JAC from JFA original 6/92. Later additions and comments by TSK in italics.
A. Modified from Drubin lab protocol
1. Culture: Either log phase or 1:100 of a stationary culture grown for 3-5 hrs (2-3 doublings.) If log phase is 5 x 106/ml, then 5 ml yields 2.5 x 107.
2. Fix by addition of 0.67 ml 37% Formalin to 5 ml culture medium (5%). Stand at RT x 1 hr.
3. Spin GP 3000 rpm x 3 min. Wash x 2 w/ 2 ml 1.2 M sorbitol, 0.1 M KPi, pH 7.5.
4. Suspend cells in 0.5 ml same buffer. Add 1 µl of b-mercaptoethanol and 20 µl of 1 mg/ml of zymolyase (stored in freezer). Incubate 30-40 min at 37°C.
5. Place 15 µl of 1 mg/ml poly-lysine (stored in freezer) on each well of multi-well slide or on circle made with rubber cement on glass coverslip. Sit for 2 min, rinse w/ water and dry.
6. Spin 500 rpm x 5 min in GP. Remove most of sup't. Suspend cells gently and place 15 µl of suspension on well. Allow to settle x 15 min. Aspirate gently.
7. Permeabilize with MeOH -20°C x 6 min, then Acetone -20°C x 30 sec, using Coplin jars in 418 freezer. Air dry thoroughly to get maximum flattening and therefore resolution of actin cables.
8. Alternative permeabilization (if Abs not tolerate MeOH/Acetone) is 0.1% Triton in PBS. Consider wash into water and air dry to flatten.
9. Wash each well x5 w/ PBS/BSA (1 mg/ml). Incubate w/ 15 µl primary Ab in PBS/BSA x 1 hr.
10. Repeat 9, except use secondary Ab. Note Drubin likes secondary's that are not affinity-purified, for maximum signal.
11. Wash w/ PBS x5 and mount w/ phenylene diamine or n-propylgallate mounting medium.
Zymolyase sol'n: Zymolyase-20T (Arthrobacter luteus), ICN Biochemicals, Cat # 32-092. 1 mg/ml in 0.1 M KPi, pH 7.5, stored at -20°C (perhaps it should be frozen and NOT fresh?). Note that this concentration is more than 10x less than what was in our previous method. Probable reason why anti-actin works much better w/ this method.
Mounting medium: 50% glycerol in PBS, plus 1 mg/ml of either p-phenylene diamine or n-propyl gallate. The latter needs warming and vortexing to dissolve. We often have a working tube of this in the refrigerator.
(This protocol worked better for anti-Cap1/2p staining, than original Drubin protocol.)
Growth and Fixation
a. Grow the appropriate yeast strain at 30ûC (or the appropriate temperature for ts strains) to a concentration of 4-8 x 106 cells/ml. This cell density corresponds to A600 =0.3-0.6, based on the general conversion of 13 x 106 cells/ml for A600 = 1. It is important not to grow the cells to high density (>2 x 107 is probably too high), otherwise cables are not present. Need 10 x 106 cells for one staining, therefore 2 ml is sufficient. Larger cultures are also convenient because the fixed cells can be stored and then stained later.
Dilute stationary culture 1:100 in 5 ml of YPD, grow overnight. In the morning you will get OD600 = 0.8-1. Dilute in 5 ml 1:5, in 3-4 hr you will get 0.4-0.6, ready to use. TSK.
b. When cells have reached the proper density, fix by adding formaldehyde directly to the culture medium to a final concentration of 2-4 %. We use the commercial 37% liquid stock for this application. JFA showed that formaldehyde prepared fresh from paraformaldehyde does not give better results. ("Purists" insist on fresh.) Incubate 10 min at room temperature with gentle agitation.
It is better to add 1/5 of the volume of 1:1 solution of formaldehyde and 1M KPi (pH 7.0). TSK.
c. Harvest the cells by spinning in tabletop at 3000 rpm (2000 gmax) x 5 min and resuspend in 1-2 volumes of 0.1 M KPi pH 7 containing 2-4% formaldehyde. Incubate 1 hr at room temperature with gentle agitation. The longer you fix, the more resistant the cells become to zymolyase treatment .TSK.
d. Optional:Harvest the cells and resuspend in 1-2 volumes of 0.1 M KPi pH 7 containing 10 mM ethanolamine to quench residual formaldehyde. Incubate 10 mins., room temperature. Resuspend the cells in 0.1 M KPi. Sonicate briefly (For 30 ml: setting 3, microprobe, 20 sec per DG) to disperse clumps (if the strain forms clumps). Spin down and resuspend in 0.1 M KPi in about 1/30th of the original culture volume. Cells can be stored at 4ûC and used for 2-3 days.
After formaldehyde treatment , wash twice in KS buffer, then resuspend in 1/10 of the V of KS. TSK.
Staining
1. Harvest ~107 fixed cells in the microfuge. Suspend cells in 1 ml Zymolyase sol'n.(To 1 ml of culture 50 mkl of zymolyase (10 mg/ml) + 1.75 merkaptoethanol). Incubate at 37ûC with very gentle agitation, just sufficient to keep cells in suspension.
2. Monitor digestion every 15 minutes by placing 5 µl on a slide with 5 µl of either KS or 10% Triton in water. Look for two things: First, in KS the cells should be intact, namely refractile by phase contrast. Lysed cells implies that agitation is too aggressive. Second, cells in Triton will be phase dark when Zymolyase has been sufficient to permit lysis. Stop Zymolyase digestion at this point.
Incubation at 370 until cells become barely visible in bright field and black in polarized. The time depends on culture stage, time of fixation and culture genotype. Varies between 25-35 min (Gena), 75 min (TSK) , 45 min (JFA).TSK
3. Wash the cells x 2 with KS. Spin 1000 rpm (250 gmax) for 5 min in tabletop. Suspend very gently with Pasteur pipet. Suspend in 100 µl KS at end. Washing from zymolyase is very important for Triton permeabilization, but can be skipped off in case of methanol/acetone treatment. TSK.
4. Place cell suspension in center of 18 mm polylysine-coated coverslip or on single well of polylysine-coated Flow multi-well slide (the former gives better optics but is more tedious to use). Incubate 5 min, rinse gently w/ water, and air dry.
Rinsing with water helps a lot to remove the remnants of sorbitol at the bottom of the well and to flatten the cells. Air dry will not be successful, if sorbitol remains at the bottom of the well. TSK.
5. This step is optional depending on the antibody. For some antibodies, skip to step 6.
D. Drubin's anti-actin antibody needs treatment of the cells w/ methanol, presumably to denature the actin, to work. Place the coverslip in methanol at -20° for 6 min. A Coplin jar (for coverslips or slides) sitting in the freezer works well. Then blot briefly and place in acetone at -20° for 30 sec. Air dry.
For Cap2/1p it is better to permeabilize cells with 0.1% Triton/PBS for 30 min, and rinse them very carefully, three times, in PBS afterwards. This can be done in the wells, but then a very small amount of Triton solution and water (5µ) should be added, as it tends to spread around the well.TSK.
6. Incubate w/ blocking sol'n for 20 min at RT.
7. Remove and add primary antibody in blocking solution for 1 hr at RT. A good affinity-purified polyclonal or monoclonal should work at 1 µg/ml. Both less and more, up to 100 µg/ml should be considered and tested if necessary. Primary antibody must be obtained against total, non-denatured CAPp of yeast and affinity-purified, as well as the next antibodies. For capping protein, and possibly for other proteins, less abundant that actin, the primarily antibody must be pre-adsorbed towards the yeast cells with disruption for the relevant protein. TSK.
As a positive control, use anti-tubulin mAb from L. Goldstein, found in cold room antibody box. Dilute 1:10. Possible negative controls: 1) non-immune immunoglobulins from the same animal, 2) no primary Ab, 3) immune primary antibody absorbed against the antigen.
8. Rinse x 3 w/ blocking solution. ?JFA 10 times. Be gentle to avoid washing the cells of the surface. Aspirate at one corner, then add slowly at another corner.
9. Consider sandwich antibody to amplify signal at this point. i.e. JFA used 1 µg/ml rabbit anti-CP, then 1 µg/ml goat anti-rabbit, then 1 µg/ml DTAF donkey anti-goat. Additional amplification was obtained with rabbit anti-goat, and then one can do goat anti-rabbit and rabbit anti-goat indefinitely.
10. For second antibody, use fluorescent-conjugated antibody to the first antibody. We have a variety of affinity-purified antibodies, which are used at various dilutions. One can check the product literature from the company (antibody notebook), use 1 µg/ml, or ask someone in the lab. Consider adding fluorescent phalloidin to this solution, for a double-stain. Background can be reduced if fluorescent-conjugated antibody is absorbed against the yeast cells.TSK.
11. Incubate 1 hr at RT. Wash x 3 (?5) w/ PBS.
12. Mount by placing 10 µl of mounting medium on a slide and inverting the 18 mm coverslip on it. Remove excess liquid by gentle aspiration (don't slide the coverslip around on the slide), dry the edges and seal w/ nail polish (Revlon Creme #77 or 76). For the multi-well add 10 µl to each well, remove excess liquid by filter paper and aspiration.TSK.
Buffers and Reagents. Want Azide in Everything but Remember Azide Kills GFP.
KS: 0.1 M KPi pH 7, 1 M Sorbitol.
Zymolyase Sol'n: Zymolyase at 10 U per ml in KPi/Sorbitol with 25 mM 2-mercaptoethanol (1.75 µl neat per ml). Currently use Zymolyase 20T (20,000 U per g). Make stock of 10 mg per ml, then add 50 µl of stock per ml of final solution.
PBS or TBS. From a 10 X stock.
For 1 liter of 10X PBS: 80g NaCl;2g KCl; 2g KH2PO4;11.5g Na2HPO4 anhydrous, or 21.67 of Na2HPO4 x 7 H2O; 1g NaN3;
Mounting medium: 50% glycerol in PBS, plus 1 mg/ml of either p-phenylene diamine or n-propyl gallate. The latter needs warming and vortexing to dissolve. We often have a working tube of this in the refrigerator.
Polylysine coating: 1 mg/ml poly(L)lysine hydrobromide (>300K) in water, which is stored in -20° chest freezer. Make the solution 1:2000 in PhotoFlo by diluting a 10% stock by 200-fold. Apply a thin film to a coverslip (?cleaned). ?Air dry in oven. Rinse w/ water and air dry in oven again.
Blocking sol'n. PBS or TBS (Tris-buffered saline) with any or all of the following:
0.01% Tween-20 or Triton X-100 (note that this detergent may be essential if you have not done the methanol/acetone extraction, which dissolves the plasma membrane lipids and thereby permeabilized the cell.)
1% nonfat dry milk
1% fish gelatin
1% BSA
Consider that one or another of these proteins may affect the signal produced by the antibody as well as lower the noise due to non-specific staining.
For actin staining BSA is enough, for capping protein it is better to use all the three reagents.TSK.
Meth.Enzymol. 1991. V.194. P.644-661. (p.651).
1ml of yeast cells (cap-null) grown to stationary phase and fixed in formaldehyde+KPi buffer like for staining. Washed in KS and tereated for 0.5 hr in Zymolyase/merkaptoethanol soln. Washed thoroughly (three times) in KS - important, as this extract may contain proteases. Resuspended in 200 mkl BSA/PBS + antibody (10x of working concentration). After incubation in microfuge tube for 1 hr on rotary shaker spin down gently, collect the supernatant, then add 200 mkl of PBS/BSA to the pellet, mix, spin down gently. Combine both supernatants and spin down vigorously for 15 min at 15 000 . Keep at 40. This should be the working solution , applied without dilution.TSK.