- Blocking reagent, 5%. Dissolve 2.5 g of BSA in 50 ml PBS.
ΔCRITICAL Make fresh and dissolve slowly in a rotator.
Glucose (for GLOX buffer) 0.25 g/ml. Dissolve 2.5 g in 10 ml PBS. Filter-sterilize and store at 4⁰C.
Glucose 500 mM Dissolve 4.5 g of Glucose in 50 ml ddH2O. Filter-sterilize and store at 4⁰C.
Glucose oxidase 3 mg/ml. Dissolve 5 mg of glucose oxidase (60% purity) in 1 ml PBS. ΔCRITICAL Use fresh, dissolve only before use.
EGTA 500 mM Dissolve 9.51 g of - EGTA in 50 ml ddH2O, add drops of NaOH to help dissolve EGTA, which will only dissolve at pH 8.
PBS (10 mM phosphate buffer, 2.7 mM KCl, 137 mM NaCl). Mix 5 PBS tablets in 1 l of ddH2O, pH 7.4. Filter-sterilize the solution and store it at RT for up to 6 months.
PBS++ (10 mM phosphate buffer, 2.7 mM KCl, 137 mM NaCl, 1 mM CaCl2, 0.5 mM MgCl2). Mix 5 PBS tablets, 110,98 mg CaCl2 and 47,6 mg MgCl2 in 1 l of ddH2O, pH 7.4. Filter-sterilize the solution and store it at RT for up to 6 months.
Gelatin solution for coating. Dissolve Gelatin to a concentration of 0.5% (w/v) in PBS. Autoclave and aliquot in filter-sterilized 50 ml aliquots. Store at 4⁰C for up to 6 months.
Paraformaldehyde solution. 20% (w/v) stock solutions.27 !CAUTION Wear appropriate protective safety equipment and work under a fume hood. Weigh 20 g of PFA inside the fume-hood and dissolve in ~50 ml of ddH2O. Stir continuously the mixture slowly on a hot plate keeping the temperature below 55-57⁰C. If necessary add drops of NaOH until all PFA is dissolved. Once the solution does not look cloudy anymore, PFA is dissolved and HCl should be used adjust the pH to 7.2. Let the solution cool down and adjust the final volume to 100 ml with ddH2O. Store in filter-sterilized 5ml aliquots at -20⁰C for up to a year.
ΔCRITICAL thaw the solution only before use.
ΔCRITICAL PFA 20% stock solutions must be prepared from powder PFA.
PIPES 500 mM pH 6.8. Dissolve 15.12 g of PIPES in 50 ml ddH2O. Check pH and, if necessary, adjust to pH 6.8. Adjust final volume to 100 ml. Filter-sterilize. Store in aliquots at -20⁰C.
MEA 1 M. Dissolve 0.61 g MEA in 5 ml PBS, adjust pH with NaOH to pH 8. Adjust volume to 10 ml. Stock aliquots at -20⁰C for up to a year.
!CAUTION Wear appropriate protective safety equipment and work under a fume hood.
MES 100 mM Dissolve 0.98 g of MES buffer in 25 ml ddH2O, adjust pH to 6.1, add ddH2O up to 50 ml. Filter-sterilize and store at 4⁰C.
MgCl2 50 mM Dissolve 48 mg of MgCl2 in 10 ml. Filter-sterilize and store at RT.
NaBH4 0.1% (w/v) Dissolve 0.01 g of NaBH4 in 10 ml PBS.
!CAUTION Wear appropriate protective safety equipment and work under a fume hood.
ΔCRITICAL Prepare fresh before use.
- NaCl 4 M Dissolve 11.7 g of NaCl in 50 ml ddH2O. Filter-sterilize and store at RT.
ΔCRITICAL Filter sterilize all reagents. All solutions used before fixation of the cells must be sterile as bacterial toxins can induce changes in the actin cytoskeleton. Solutions used after fixation must be sterile to avoid background.
Preparation coated coverslips. • TIMING ~1-2 h
1| Place one coverslip of 24 mm in each well in a 6-well cell culture plate, wash coverslips for 5 minutes with 2 ml 70% EtOH, rinse twice with 2 ml PBS. Coat wells with 1-2 ml of 0.5% gelatin solution in a humidified incubator at 37⁰C for 30 minutes. Rinse twice with PBS++.
ΔCRITICAL Coverslips should be coated with gelatin just before plating the cells.
Cell seeding and growth. • TIMING ~48 h
2| Two days before the imaging, seed 150.000 HeLa cells (or 100.000 COS-7 cells) per well on freshly gelatin-coated coverslips.
ΔCRITICAL STEP Control: Seed 2 replicas per condition. One of the replicas will be used as a control of the fixation effect on the actin-binding protein to be studied.
3| The day before the imaging, change the growing medium of the cells to DMEM supplemented with 0.1% FCS. Leave cells in this medium for ~16 hours.
4| On the day of the imaging, stimulate cells with agonist of interest at the desired concentration.
ΔCRITICAL After a few minutes (exact time depends on the specific agonist and should be determined through time-course experiments) observe the cells under a phase contrast microscope to confirm that they have responded to the agonist. Cells should show agonist-specific phenotype.
Fixation. • TIMING ~30 min
!CAUTION For the preparation and use of the fixation solutions, wear all the time appropriate safety equipment and work under a fume-hood.
ΔCRITICAL When changing buffers, pipet gently and never let the sample dry out for maintenance of the cytoskeletal structures and to avoid background.
5| Prepare the appropriate fixation solution and fix cells.
(A) Glutaraldehyde fixation and quenching 11
!CAUTION Glutaraldehyde fixation is the recommended fixation method for the preservation of the actin cytoskeleton architecture, but it is often incompatible with antibody-based detection of actin-binding proteins.
i. Warm up PBS++ to 37⁰C in a waterbath
ii. Prepare two aliquots of cytoskeleton buffer (10 mM MES, 150 mM NaCl, 5 mM EGTA, 5 mM Glucose, 5 mM MgCl2) by mixing stock solutions: 1 ml MES (100 mM), 375 µl NaCl (4 M), 100 µl EGTA (500 mM), 100 µl Glucose (500 mM), 1 ml MgCl2 (50 mM) and ddH2O up to 10 ml
ΔCRITICAL Buffer must be freshly prepared
iii. Prepare the first fixation buffer by adding 0.3% glutaraldehyde and 0.25% Triton X-100 to one 10 ml aliquot of cytoskeleton buffer. Prepare the second fixation buffer by adding 0.5% glutaraldehyde to one 10 ml aliquot of cytoskeleton buffer.
!CAUTION When using glutaraldehyde work always under the fume hood.
iv. Remove the growing medium and rinse cells carefully with 2 ml of warm PBS++.
v. Remove PBS++ and replace it with the first fixation buffer. Leave the sample fixing for 2 minutes.
ΔCRITICAL Longer fixation times can increase membrane permeability and result in the loss of soluble proteins.
vi. Remove the first fixation buffer and add 1 ml of the second fixation buffer for 8 minutes.
ΔCRITICAL Longer fixation times can increase the background and make epitopes unavailable to the antibodies.
vii. Rinse twice with 2 ml of PBS.
viii. Remove PBS and add 0.1% NaBH4 solution for 10 minutes.
!CAUTION Perform all NaBH4 involving steps under a fume hood with proper protection
ΔCRITICAL NaBH4 solution must be freshly prepared.
ΔCRITICAL After a few minutes of incubation bubbles should appear around the coverslip.
ix. Rinse twice with 2 ml of PBS for 5 minutes.
(B) Paraformaldehyde fixation and permeabilization
ΔCRITICAL STEP Fixation should occur at the correct temperature for the appropriate time to preserve the architecture of the actin cytoskeleton. Do not exceed fixation time and optimal temperature. Perform at 37⁰C until fixation is complete
i. Prepare PFA in stabilization buffer (PEM: 80 mM PIPES pH6.8, 5 mM EGTA, 2 mM MgCl2, 4% PFA) by mixing stock solutions: 4 ml PIPES (500 mM), 0.25 ml EGTA (500 mM), 1 ml MgCl2 (50 mM), 5 ml PFA (20 %) and ddH2O up to 25 ml.
ΔCRITICAL PFA must be freshly thawed.
ii. Warm up PBS++ and PFA in cytoskeleton buffer to 37⁰C in a waterbath.
ΔCRITICAL Both buffers must be warm before fixing the cells.
iii. Remove the growing medium and rinse cells carefully with 2 ml warm PBS++.
iv. Remove PBS++ and replace it with PFA in PEM buffer. Leave the sample fixing for 10 minutes.
ΔCRITICAL STEP Longer fixation times can increase the background when imaging and mask epitopes.
v. Rinse twice with 2 ml of PBS.
vi. Remove the fixation solution and add 2 ml of 0.5% Triton in PBS for 10 minutes.
vii. Rinse twice with 2 ml of PBS for 5 minutes.
6| Check the quality of the fixation. Cells should still be visible in a phase contrast microscope. In case some proteins are genetically tagged with a fluorescent protein (FP), verify that these still have the expected localization with a fluorescent microscope.
Paraformaldehyde and Glutaraldehyde blocking. • TIMING > 1 hour
ΔCRITICAL Blocking solution must be prepared freshly.
7| Replace PBS from sample with blocking 5% BSA solution. Allow BSA to block for at least 1 hour. Alternatively, samples can be blocked at 4⁰C overnight.
ΔCRITICAL STEP Efficient blocking is crucial for super-resolution imaging to prevent unspecific binding of antibodies.
□PAUSE POINT Samples can be stored at this point at 4⁰C for up to 1 week.
Immunofluorescence staining for super-resolution imaging. • TIMING ~2 hours
8| Primary antibody staining (if only Phalloidin staining is to be done jump to step 9|)
ΔCRITICAL STEP Control Leave one of the two replicas without primary antibody. This coverslip will serve as a control for the effects of the fixation on the protein of interest as well as the specificity of the primary antibody.
i. Dissolve antibody in 100 µl of 5% BSA at a concentration determined before in optimization of primary antibody labelling for the specific protein/antibody combination
ii. Pick the coverslip with forceps and incubate it facing down in a drop of 100 µl of antibody solution.
iii. Incubate the sample for 45 minutes at room temperature
ΔCRITICAL Longer incubation times will increase unspecific binding.
iv. Rinse twice with PBS
9| Secondary antibody staining/Phalloidin staining
ΔCRITICAL Efficient blinking is crucial for super-resolution imaging of challenging structures such as actin filaments, which are very dense in cells and small in diameter. Therefore, we recommend Phalloidin to be labelled with AlexaFluor-647, the best dye for stochastic SMLM. As Phalloidin has a finite off-rate, for best results it must be imaged within a few hours of staining.
ΔCRITICAL STEP Control samples (i.e. the coverslips without primary antibody) can be labelled here only with the secondary antibody to test the effect of the fixation procedure on the labelling of the actin-binding protein.
i. Prepare a solution in 100 µl of 5% BSA with 0.6 U of Phalloidin (Molecular probes for Life Technologies) and 10 µg/ml of secondary antibody
ii. Pick the coverslip with forceps and incubate facing down in a drop of 100 µl of staining solution.
iii. Incubate the sample for 30 minutes at room temperature always protected from light.
ΔCRITICAL Longer incubation times will increase the unspecific binding.
iv. Rinse twice with PBS
10| Check the quality of the staining. Fluorescence of AlexaFluor-532 should be visible in a standard epi-fluorescence microscope. For proteins genetically tagged with a FP, verify that they are still localized where expected and so should the proteins stained with antibodies.
Sample mounting and super-resolution imaging. • TIMING ~1hour
11| Prepare the imaging buffer (10% Glucose, 0.5 mg/ml Glucose Oxidase, 40 µg/ml Catalase, 100 mM MEA in PBS) by mixing 200 µl glucose (prepared previously at 0.25 g/ml for GLOX buffer), 83 µl Glucose Oxidase, 50 µl MEA, 6.75 µl Catalase and PBS up to 500 µl. As the concentration of the stock solution of Catalase shows batch-to-batch differences, we add 6.75 µl of a 1:10 dilution of our concentrated Catalase corresponding to a final concentration of 40 µg/ml.
ΔCRITICAL STEP Imaging buffer must be prepared freshly, Glucose Oxidase freshly prepared, and MEA thawed on the day of imaging.
ΔCRITICAL As efficient blinking is crucial for super-resolution imaging, the buffer used must be selected and optimized for each fluorophore. For actin imaging, we recommend the use of AlexaFluor-647 with a Glucose Oxidase-based oxygen-scavenging system.
ΔCRITICAL, In order to preserve the integrity of the cells and the actin cytoskeleton, it is crucial to pipet gently and to maintain the sample always wet when changing buffers.
12| Mount the sample with the forceps on an open imaging ring.
i. Clean the bottom of the coverslip thoroughly with 70% ethanol. Wipe carefully in the same direction to avoid smudges. Let dry.
ii. Add imaging buffer (500 µl)
iii. Clean the objective with a 50-50 petroleum ether – butanol solution with a lens paper and always in the same direction. Let dry.
iv. Let the sample holder stabilize for ~5 minutes before imaging.
13| Image the sample with the microscope. (Ref: Leica SR GSD 3D).
ΔCRITICAL STEP Super-resolution imaging is not a straightforward task and should be performed preferably by an expert user. Detailed protocols and troubleshooting for imaging procedure are available elsewhere 23.
i. Select the desired imaging setting, to image close to the basal membrane use the TIRF option, and for actin in the cytoplasm or ruffles choose the EPI fluorescence option.
ii. Select the cell you want to image.
iii. In the super-resolution mode, excite cells with the appropriate laser/filter combination at high laser power in EPI mode to transfer molecules to the dark state. Depending on the fluorophore used and the density of the molecule, the time required to transfer molecules to the dark state will change.
Once molecules have been transferred to the dark state, select the appropriate imaging method (TIRF or EPI) and image for the necessary time. Depending on the fluorophore used and the labelling density, the time required to obtain an image will change.
14| (Optional) Perform data analysis with (open source) packages like Thunderstorm 23 to locate the centre of the blinks and create a final SRM image. In some cases, structured background subtraction with a temporal median filter can be beneficial 28.
ΔCRITICAL STEP Compare the results of the negative control sample with the samples stained also with primary antibody (Figure 2). If the pattern is the same, it is possible that the proteins (or the epitopes) of interest were lost (or masked) during fixation. Alternatively, the primary antibody may not be specific.