Name: visualizing the actin and microtubule cytoskeletons at the b-cell immune synapse using stimulated emission depletion (sted) microscopy
Uploaded: 2018-04-09T14:00:00
Description: Watch this Scientific Journal Video about Visualizing the Actin and Microtubule Cytoskeletons at the B-cell Immune Synapse Using Stimulated Emission Depletion STED Microscopy at JoVE.com

We thank the UBC Life Sciences Institute (LSI) Imaging Facility for supporting and maintaining the STED microscope. This work was funded by grant #68865 from the Canadian Institutes of Health Research (to M.R.G.). We thank Dr. Kozo Kaibuchi (Nagoya University, Nagoya, Japan) for the CLIP-170-GFP plasmid.

All animal procedures were approved by the University of British Columbia Animal Care Committee.
1. Expressing GFP-fusion proteins in A20 B-lymphoma cells
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	<li>Culture A20 cells in complete RPMI medium (RPMI-1640 supplemented with 10% heat-inactivated fetal bovine serum (FBS), 50 &#181;M 2-mercaptoethanol, 2 mM glutamine, 1 mM pyruvate, 50 U/mL penicillin, and 50 &#181;g/mL streptomycin) at 37 &#176;C in a tissue culture incubator with 5% CO2.</li>
	<li>Prepare the transfecti...

<ol>
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Detailed images of cytoskeletal structures can be obtained using STED super-resolution microscopy, which can theoretically achieve a resolution of 50 nm, compared to conventional confocal microscopy, for which the diffraction-limited resolution is ~200 nm24. The ability to resolve finer structures is further enhanced by using deconvolution software to calculate the most likely position of the original light source from the observed &#34;blurred&#34; fluorescence signal. This protocol describes met...

When B cells bind to polarized arrays of antigens (e.g., displayed on the surface of antigen-presenting cells (APCs)), the resulting B-cell receptor (BCR) signaling drives the formation of a classic immune synapse structure, which was first described in T cells1,2,3,4,5,6,7,8,9,10,11,12,13. Initially, microclusters of antigen-bound BCRs form at the periphery of the B cell:APC contact site. These microclusters then move towards the center of the antigen contact site, where they coalesce into a central supramolecular activation cluster (cSMAC) that forms the core of the immune synapse. Immune synapse formation optimizes BCR signaling and facilitates BCR-mediated antigen extraction from the APC membrane14. This antigen acquisition, which is followed by BCR-mediated antigen internalization and subsequent antigen processing, allows B cells to present peptide:MHC II complexes to T cells and elicit T cell help14. Because immune synapse formation promotes B cell activation, elucidating the mechanisms that establish this functional pattern of receptor organization can provide new insights into how humoral immune responses are initiated and regulated.
Reorganization of both the actin and microtubule cytoskeletons is essential for immune synapse formation. Localized BCR signaling stimulated by a spatially-polarized array of antigens induces rapid and dramatic remodeling of the actin cytoskeleton1,15. The formation of dendritic actin structures at the periphery of the B cell exerts pushing forces on the plasma membrane and promotes B cell spreading. This allows the B cell to scan a greater area of the antigen-bearing surface, and increases the number of BCRs that bind antigen and activate BCR signaling pathways. At the same time, the MTOC and the microtubule network are reoriented towards the site of antigen contact. As the MTOC approaches the antigen contact site, microtubules emanating from the MTOC extend along the inner face of the plasma membrane at the interface between the B cell and the antigen-bearing surface16,17. These juxtamembrane microtubules can then act as tracks for the dynein-mediated centripetal movement of antigen-bound BCR microclusters18, leading to the formation of a cSMAC.
The reorientation and polarization of the MTOC towards the immune synapse requires intact actin and microtubule cytoskeletons, and often depends on interactions between the cortical actin network and microtubules16,17,19,20. Cortical actin-binding proteins, such as IQGAP1, can capture microtubules by interacting with protein complexes that decorate the microtubule plus-ends21. These dynamic complexes of plus-end binding proteins include EB1 and CLIP-170, which are collectively referred to as microtubule plus-end tracking proteins (+TIPs)21,22. +TIPs at the ends of microtubules can bind to proteins that are associated with either the plasma membrane or the cortical actin cytoskeleton. This allows force-generating mechanisms (e.g., the minus-end directed movement of cortically-anchored dynein along microtubules) to exert pulling forces on microtubules, and thereby reposition the MTOC. CLIP-170 can bind to the actin-associated scaffolding protein IQGAP123, and we have shown that both of these proteins are required for BCR-induced MTOC polarization towards the immune synapse17. This IQGAP1-CLIP-170 interaction may play a key role in coordinating the remodeling of the actin cytoskeleton with the repositioning of the microtubule network at the B-cell immune synapse.
Conventional fluorescence microscopy has revealed the dramatic reorganization of the actin and microtubule cytoskeletons during B-cell immune synapse formation2. However, this approach cannot resolve small cellular structures in detail due to the diffraction limit of light, which, according to Abbe&#39;s law, is dependent on the wavelength of light used to illuminate the sample and the aperture of the objective24. This diffraction limit constrains the resolution of conventional light microscopes to 200-300 nm in the lateral direction and 500-700 nm in the axial direction25. Therefore, smaller subcellular structures, as well as the fine details of the actin and microtubule cytoskeletons, could only be observed using electron microscopy. Electron microscopy imaging of the cytoskeleton is time consuming, requires harsh sample fixation and preparation protocols that can alter biological structures, and is limited to antibody-mediated detection. The ability to immunostain and simultaneously image multiple proteins or cellular structures is a substantial advantage of fluorescence microscopy. Moreover, expressing fluorescent fusion proteins in cells enables real-time imaging and is useful when effective antibodies for immunostaining the protein of interest are not available.
Recent technological advances in super-resolution microscopy have overcome the diffraction limits of light and allowed the visualization of nanoscale cellular structures24. One such super-resolution microscopy technique is called stimulated emission depletion (STED) microscopy. STED employs two lasers, where one laser excites the fluorophore and a second laser with a donut-shaped pattern selectively suppresses the fluorescence emission around the fluorophore. This reduces the point-spread function (apparent area) of a single fluorescent particle and provides a sub-diffraction limit fluorescent image25,26. Ground-state depletion microscopy also employs fluorescence-based techniques to acquire super-resolution images. However, the image acquisition and reconstruction times are long, there are only a limited number of fluorophores that can be used, and the simultaneous high-resolution imaging of multiple cytoskeletal components is technically challenging because maintaining actin and microtubule structures requires different fixation procedures. Therefore, STED has multiple advantages over electron microscopy and other super-resolution microscopy approaches in that it offers rapid image acquisition, has minimal post-processing requirements, and employs the same fluorophores and staining techniques that are used for conventional fluorescence microscopy of fixed samples26.
Super-resolution microscopy has now been used to visualize actin structures at the immune synapse in natural killer (NK) cells and T cells26,27,28,29,30,31. However, super-resolution imaging of the microtubule cytoskeleton, as well as the coordinated reorganization of the actin and microtubule cytoskeletons during immune synapse formation, has only recently been reported17. We used STED microscopy to image B cells that had been allowed to spread on coverslips coated with anti-immunoglobulin (anti-Ig) antibodies, which stimulate BCR signaling and initiate cytoskeleton reorganization. When plated on immobilized anti-Ig antibodies, B cells undergo dramatic actin-dependent spreading, which recapitulates the initial events during immune synapse formation. Importantly, STED microscopy revealed the fine details of the dendritic ring of F-actin that forms at the periphery of the immune synapse and showed that the MTOC, as well as the microtubules attached to it, had moved close to the antigen contact site17. These microtubules extended outward towards the peripheral ring of F-actin. Moreover, multi-color STED imaging of various combinations of F-actin, tubulin, IQGAP1, and GFP-tagged CLIP-170 +TIPs showed that microtubule plus-ends marked by CLIP-170-GFP were closely associated with the peripheral actin meshwork and with IQGAP1, a cortical capture protein17.
Here, we present a detailed protocol for imaging the actin and microtubule cytoskeletons at the immune synapse using STED microscopy. These methods have been optimized using the A20 murine B cell line, which has been widely employed for studying BCR signaling and immune synapse formation17,32,33,34,35,36,37,38,39. Because commercial antibodies to CLIP-170 did not work well for immunostaining in previous experiments, we describe in detail the expression of GFP-tagged CLIP-170 in A20 cells, along with staining protocols for simultaneously visualizing up to three cytoskeletal components or cytoskeleton-associated proteins. Methods for using STED microscopy to image actin at NK cell immune synapses have been described previously40. Here, we extend this to acquiring multi-color super-resolution images of both the actin and microtubule cytoskeletons in B cells.
A critical consideration for super-resolution microscopy is using the appropriate fixation procedures for maintaining cellular structures and preventing damage to fluorescent proteins. The fixation and staining methods presented herein have been optimized to retain GFP fluorescence and provide high-resolution imaging of the actin and microtubule networks. When expressing fluorescent proteins, it should be noted that B cells are usually difficult to transfect. Using this protocol, 20-50% of A20 cells typically express the transfected GFP fusion protein, and among this population the levels of protein expression are variable. Nevertheless, super-resolution imaging of actin and microtubules using the procedures we describe is quite robust and high-quality images are readily obtained. Despite their small size relative to A20 cells, we show that these procedures can also be used to image the microtubule network in primary B cells that have been briefly activated with lipopolysaccharide (LPS). We have shown that LPS-activated primary B cells can be transfected with siRNAs at relatively high efficiency (i.e., such that protein depletion can be detected by immunoblotting), making them a good alternative to the use of B cell lines for some studies17.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Cell culture</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>A20 mouse B-lymphoma cells</td>
			<td>ATCC</td>
			<td>TIB-208</td>
			<td>Murine B-cell lymphoma of Balb/c origin that expresses an IgG-containing BCR on its surface</td>
		</tr>
		<tr>
			<td>RPMI-1640</td>
			<td>Thermo Fisher Scientific</td>
			<td>&#160;R0883</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum</td>
			<td>Thermo Fisher Scientific</td>
			<td>12483020</td>
			<td>Heat inactivate at 56 oC for 30 min</td>
		</tr>
		<tr>
			<td>2-mercaptoethanol</td>
			<td>Millipore Sigma</td>
			<td>M3148</td>
			<td></td>
		</tr>
		<tr>
			<td>Glutamine</td>
			<td>Millipore Sigma</td>
			<td>G5763</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium pyruvate</td>
			<td>Millipore Sigma</td>
			<td>P5280</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin</td>
			<td>Thermo Fisher Scientific</td>
			<td>15140122</td>
			<td>Liquid, 10,000 units</td>
		</tr>
		<tr>
			<td>Additional materials for primary B cells</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>70-&#181;m cell strainer</td>
			<td>Corning</td>
			<td>352350</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnetic bead-based B cell isolation kit</td>
			<td>Stemcell Technologies</td>
			<td>19854</td>
			<td>EasySep Mouse B cell Isolation kit</td>
		</tr>
		<tr>
			<td>Lipopolysaccharide (LPS)</td>
			<td>Millipore Sigma</td>
			<td>L4391</td>
			<td>LPS from E. coli 0111:B4</td>
		</tr>
		<tr>
			<td>B cell-activating factor (BAFF)</td>
			<td>R&#38;D Systems</td>
			<td>2106-BF-010</td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Transfection</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Plasmid encoding CLIP-170-GFP</td>
			<td></td>
			<td></td>
			<td>Gift from Dr. Kozo Kaibuchi (Nagoya Univ., Nagoya, Japan); described in Fukata et al. Cell 109:873-885, 2002</td>
		</tr>
		<tr>
			<td>Recommended transfection reagents and equipment </td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Amaxa Nucleofection kit V</td>
			<td>Lonza</td>
			<td>VCA-1003</td>
			<td>Follow the manufacturer&#39;s directions for mixing the transfection reagents with the DNA</td>
		</tr>
		<tr>
			<td>Amaxa Nucleofector model 2b</td>
			<td>Lonza</td>
			<td>AAB-1001</td>
			<td>Program L-013 used</td>
		</tr>
		<tr>
			<td>Falcon 6-well plates, TC treated, sterile</td>
			<td>Corning</td>
			<td>353046</td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Coating coverslips</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>18-mm diameter round #1.5 cover glasses</td>
			<td>Thomas Scientific</td>
			<td>1217N81</td>
			<td>Similar product: Marienfield-Superior catalogue #117580</td>
		</tr>
		<tr>
			<td>Forceps</td>
			<td>Fisher Scientific</td>
			<td>1381242</td>
			<td>Dissecting extra fine splinter forceps</td>
		</tr>
		<tr>
			<td>Falcon 12-well sterile tissue polystyrene tissue culture plate</td>
			<td>Corning</td>
			<td>353043</td>
			<td></td>
		</tr>
		<tr>
			<td>100% methanol</td>
			<td>Fisher Scientific</td>
			<td>A412-4</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile phosphate buffered saline (PBS) without calcium or magnesium</td>
			<td>Thermo Fisher Scientific</td>
			<td>10010049</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat-anti-mouse IgG antibody</td>
			<td>Jackson ImmunoResearch</td>
			<td>115-005-008</td>
			<td>For A20 cells</td>
		</tr>
		<tr>
			<td>Goat-anti-mouse IgM antibody</td>
			<td>Jackson ImmunoResearch</td>
			<td>115-005-020</td>
			<td>For primary mouse B cells</td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Staining</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde (16% stock solution)</td>
			<td>Electron Microscopy Sciences</td>
			<td>15710</td>
			<td>Dilute with PBS to working concentration</td>
		</tr>
		<tr>
			<td>Glutaraldehyde (50% stock solution)</td>
			<td>Millipore Sigma</td>
			<td>340855</td>
			<td>Dilute with PBS to working concentration</td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Fisher Scientific</td>
			<td>BP151-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Saponin</td>
			<td>Millipore Sigma</td>
			<td>S2149</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine serum albumin (BSA), Fraction V</td>
			<td>Millipore Sigma</td>
			<td>10735094001</td>
			<td></td>
		</tr>
		<tr>
			<td>Rabbit anti-tubulin antibody</td>
			<td>Abcam</td>
			<td>ab52866</td>
			<td>1:250 dilution recommended but should be optimized</td>
		</tr>
		<tr>
			<td>Alexa Fluor 532-conjugated goat anti-rabbit IgG</td>
			<td>Thermo Fisher Scientific</td>
			<td>A11009</td>
			<td>1:100 dilution recommended but should be optimized</td>
		</tr>
		<tr>
			<td>Alexa Fluor 568-conjugated phalloidin</td>
			<td>Thermo Fisher Scientific</td>
			<td>A12380</td>
			<td>1:100 dilution recommneded but should be optimized</td>
		</tr>
		<tr>
			<td>Prolong Diamond Antifade Mountant</td>
			<td>Thermo Fisher Scientific</td>
			<td>P36970</td>
			<td>Without DAPI</td>
		</tr>
		<tr>
			<td>Fisherbrand Superfrost Plus Microscope Slides</td>
			<td>Fisher Scientific</td>
			<td>12-550-15</td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Materials</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Parafilm</td>
			<td>VWR</td>
			<td>P1150-2</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Millipore Sigma</td>
			<td>H3375</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Fisher Scientific</td>
			<td>BP358</td>
			<td></td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>Millipore Sigma</td>
			<td>P9333</td>
			<td></td>
		</tr>
		<tr>
			<td>CaCl2</td>
			<td>Millipore Sigma</td>
			<td>C1016</td>
			<td></td>
		</tr>
		<tr>
			<td>Na2HPO4</td>
			<td>Fisher Scientific</td>
			<td>S374-500</td>
			<td></td>
		</tr>
		<tr>
			<td>MgSO4</td>
			<td>Fisher Scientific</td>
			<td>M63</td>
			<td></td>
		</tr>
		<tr>
			<td>Dextrose</td>
			<td>Fisher Scientific</td>
			<td>D16-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Microscopy</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Leica SP8 TCS STED microscope</td>
			<td>Leica</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Huygens deconvolution software</td>
			<td>Scientific Voume Imaging</td>
			<td></td>
			<td>See https://svi.nl/HuygensProducts</td>
		</tr>
	</tbody>
</table>

visualizing the actin and microtubule cytoskeletons at the b-cell immune synapse using stimulated emission depletion (sted) microscopy

B cells that bind to membrane-bound antigens (e.g., on the surface of an antigen-presenting cell) form an immune synapse, a specialized cellular structure that optimizes B-cell receptor (BCR) signaling and BCR-mediated antigen acquisition. Both the remodeling of the actin cytoskeleton and the reorientation of the microtubule network towards the antigen contact site are essential for immune synapse formation. Remodeling of the actin cytoskeleton into a dense peripheral ring of F-actin is accompanied by polarization of the microtubule-organizing center towards the immune synapse. Microtubule plus-end binding proteins, as well as cortical plus-end capture proteins mediate physical interactions between the actin and microtubule cytoskeletons, which allow them to be reorganized in a coordinated manner. Elucidating the mechanisms that control this cytoskeletal reorganization, as well as understanding how these cytoskeletal structures shape immune synapse formation and BCR signaling, can provide new insights into B cell activation. This has been aided by the development of super-resolution microscopy approaches that reveal new details of cytoskeletal network organization. We describe here a method for using stimulated emission depletion (STED) microscopy to simultaneously image actin structures, microtubules, and transfected GFP-tagged microtubule plus-end binding proteins in B cells. To model the early events in immune synapse formation, we allow B cells to spread on coverslips coated with anti-immunoglobulin (anti-Ig) antibodies, which initiate BCR signaling and cytoskeleton remodeling. We provide step-by-step protocols for expressing GFP fusion proteins in A20 B-lymphoma cells, for anti-Ig-induced cell spreading, and for subsequent cell fixation, Immunostaining, image acquisition, and image deconvolution steps. The high-resolution images obtained using these procedures allow one to simultaneously visualize actin structures, microtubules, and the microtubule plus-end binding proteins that may link these two cytoskeletal networks.

For B cells spreading on immobilized anti-Ig, STED microscopy used in conjunction with deconvolution software provides higher resolution images of cytoskeletal structures than confocal microscopy. This is evident in Figure 1, where the F-actin network was visualized using the protocol described above. A comparison of confocal and STED super-resolution images of the same sample shows that the STED images are of higher resolution and reveal more detailed struct...

Watch this Scientific Journal Video about Visualizing the Actin and Microtubule Cytoskeletons at the B-cell Immune Synapse Using Stimulated Emission Depletion STED Microscopy at JoVE.com

Visualizing the Actin and Microtubule Cytoskeletons at the B-cell Immune Synapse Using Stimulated Emission Depletion STED Microscopy

We present a protocol for using STED microscopy to simultaneously image actin structures, microtubules, and microtubule plus-end binding proteins in B cells that have spread on coverslips coated with antibodies to the B-cell receptor, a model for the initial phase of immune synapse formation.

Visualizing the Actin and Microtubule Cytoskeletons at the B-cell Immune Synapse Using Stimulated Emission Depletion (STED) Microscopy

B cells that bind to membrane-bound antigens (e.g., on the surface of an antigen-presenting cell) form an immune synapse, a specialized cellular structure ...

The overall goal of this protocol is to stain actin and microtubule cytoskeletons at the B-cell immune synapse for stimulated emission depletion, or STED microscopy, and imaging their structure, organization in spatial relationships. This method can help answer key questions about cytoskeleton organization during B-cell activation, including the reorganization of the actin and microtubule cytoskeletons during immune synapse formation. The main advantage of multi-color STED microscopy is that it allows simultaneous imaging of the actin cytoskeleton, the microtubule network, and key cytoskeleton associated proteins at nanometer scale resolution.Although this method can provide insights into B-cell activation, it can also be applied to immune synapse formation of other immune cell types such as natural killer and T-cells. Generally, individuals new to this method will find that optimizing the microscope settings for the simultaneous imaging and multiple fluorophores using STED will require some practice. Demonstrating this procedure along with Jia and Madison will be Kate Choi and Caitlin Pritchard.Begin by centrifuging 2.5 times 10 to the 6th, A20 B-lymphoma cells per each transfection, and re-suspending the cells in 100 microliters of transfection reagent supplemented with one to 2.5 micrograms of the plasma DNA of interest. Mix the solutions gently and transfer each aliquot of cells into individual wells of a six-well plate. Adjust the volume in each well to two milliliters with 37 degrees Celsius complete medium and place the cells in a 37 degree Celsius incubator for 18 hours.The next morning, dip 1.5 18 millimeter round glass coverslips into 100%methanol and let the coverslips dry completely for about 10 minutes. Place the dried coverslips in individual wells of a 12-well tissue culture plate and add 400 microliters of goat anti-mouse immunoglobulin G antibody into the center of each coverslip so that a bubble forms at the center and spreads to the edges without dripping into the well. Incubate the coverslips at room temperature for 30 minutes.Then rinse the coverslips with one milliliter of sterile PBS per well, three times, to remove any unbound antibodies. After the last wash, store each coverslip in one milliliter of fresh, sterile PBS until cell seeding. When the transfected cells are ready, collect them in a 15 milliliter conical tube by centrifugation and re-suspend the pellets in one milliliter of modified HEPES buffered saline supplemented with FBS.After counting, dilute the cells to a two by 10 to the 5th cells per milliliter concentration and modified HEPES buffered saline plus FBS, and use forceps to transfer each coverslip onto a piece of paraffin film. Add 250 microliters of cells to each coverslip and incubate the coverslips at 37 degrees Celsius for 15 minutes in the dark to allow the cells to spread across the anti-immunoglobulin G coated surfaces. At the end of the incubation, remove the excess saline from each slide and coat each coverslip with 350 microliters of fixative, taking care that the entire surface is covered.After 10 minutes in the dark at room temperature, use a micropipette to carefully aspirate the fixative from the edge of each coverslip and wash the coverslips with 500 microliters of permeabilization and blocking buffer. Then, permeabilize and block the cells with 250 microliters of fresh permeabilization and blocking buffer for 10 minutes at room temperature in the dark. Next, remove the excess buffer and label the cells with 50 microliters of the primary antibody of interest for 30 minutes in the dark at room temperature.At the end of the incubation, wash the coverslip three times with 500 microliters of staining buffer per wash and add 50 microliters of the appropriate secondary antibody to each coverslip for 30 minutes at room temperature. After washing the secondary antibody, add 10 microliters of mounting reagent to a microscope slide and mount the coverslips onto individual microscope slides, cell side down. Then allow the slides to dry overnight in the dark for next day imaging.To image the cells with STED microscopy, first turn on the STED microscope. Activate the lasers in the epifluorescence illumination lamp and open the microscope software. Set the parameters for the STED depletion lasers and load the first sample onto the microscope.Using the eyepiece, manually focus on the sample to select a cell with a moderate GFP expression. Zoom in to the selected cell and select the region of interest to be imaged. Adjust the focus so that the XY plane of the cell that is closest to the coverslip is in focus.Then, under the Acquire tab of the software, check the Between Frames option in the Sequential Scan panel to set the image acquisition to sequential frame acquisition. Set the STED laser power for each fluorophore and use the slide bar to adjust the laser powers according to the fluorophores used in the experiment. To increase the resolution and reduce the background signal, open the Acquisition settings and use the dropdown menu to select a value greater than one to increase the line and/or frame averaging.Check the gaining option for each fluorophore and the specific values for time gaining to acquire the appropriate fluorescent signal for time gated STED and increase the STED laser power to enhance the resolution. It is critical to optimize the acquisition time gaining for the microscope sample and fluorophores being used to improve the resolution without losing too much of the fluorescent signal. Then manually acquire multiple images to ensure reproducibility and deconvolve the images using a deconvolution software package according to the manufacturer's instructions.For A20 B-lymphoma cells, spread on immobilized NT immunoglobulin antibodies. STED microscopy used in conjunction with deconvolution software provides higher resolution images of cytoskeletal structures than does confocal microscopy alone. Although deconvolution of confocal images yields a substantial improvement in the resolution of the image, deconvolved STED images provide more detailed structural information than deconvolved confocal images.Single-colored STED can also be used to acquire a three-dimensional super-resolution images of the entire B-cell cytoskeletal network. In these multi-colored STED images, staining of the peripheral ring of the dendritic actin can be observed in conjunction with microtubule staining. When using cells transfected with fluorescent fusion proteins, achieving optimal expression levels and avoiding artifacts due to over-expression are significant considerations, while too low an expression of the fluorescent fusion protein also results in poor quality images.Careful focusing, to include the entire region of interest, is also essential. For example, in this image, only parts of the microtubule network was within the focal plane closest to the coverslip, preventing a complete analysis of the sample. Once mastered, this technique can be completed in two days.One day for the sample preparation and staining and one day for the STED imaging. It's important to remember to titrate the amount of plasma to use to transfect the cells so that you get a sufficient expression of fluorescent protein for detecting during imaging while avoiding artifacts from over-expression. Following this procedure, the expression of different fluorescent fusion proteins can be induced to facilitate the localization of other proteins that regulate cytoskeletal dynamics and organization, or to visualize the sub-cellular distribution of proteins involved in other cellular processes.After watching this video, you should have a good understanding of how to use STED microscopy for the imaging of cytoskeletal structures and proteins that are involved in immune synapse formation. Don't forget that working with fixatives such as glutaraldehyde can be extremely hazardous and that precautions such as working in a chemical fume hood should always be taken when using these kinds of reagents.

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