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.
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.
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'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.
All animal procedures were approved by the University of British Columbia Animal Care Committee.
1. Expressing GFP-fusion proteins in A20 B-lymphoma cells
2. Isolating Primary Mouse B Cells and Activating Them with LPS
3. Coating Glass Coverslips with Anti-Ig Antibodies
4. B Cell Spreading on Anti-Ig-Coated Coverslips
5. Fixing and Immunostaining the Cells
6. Imaging Using the STED Microscope
NOTE: Please note that all software steps described below are specific to the microscope and software we used (see the Table of Materials). The steps and settings will need to be adjusted if imaging is performed using a different microscope/software.
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 structures of the actin cytoskeleton (Figure 1). This figure also shows that deconvolution is essential for obtaining high quality STED images in which actin filaments are more clearly defined. 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. In particular, the dendritic structure of the peripheral F-actin ring is revealed in greater detail by STED microscopy (Figure 1 and Figure 2). The microtubule network at the antigen contact site was also imaged using the sample preparation and imaging protocol described above (Figure 3). Microtubules originate from a central point, which is the MTOC, and emanate outwards towards the periphery of the cell. In this experiment, the B cells were allowed to spread on anti-Ig-coated coverslips for 15 min (Figure 3), a time point at which the MTOC has moved towards the antigen contact site17. CLIP-170-GFP clusters that mark the plus-ends of microtubules can be seen at the ends of the microtubules shown in Figure 3. When the sample preparation and STED imaging of the microtubule network is optimal, continuous and distinct microtubules are observed, with CLIP-170-GFP localized either along the microtubules or at the plus-ends (Figure 3A). Sub-optimal microtubule staining, which was observed when using lower concentrations of staining antibodies or batches of α-tubulin antibodies that are more than one year old, results in microtubules appearing as discontinuous sections that are poorly resolved upon deconvolution (Figure 3B; see also Figure 5C). Although all of the CLIP-170-GFP fluorescence in these images is associated with α-tubulin-immunostained structures, one cannot distinguish whether CLIP-170-GFP is located at the plus ends, or along the length of the microtubules, due to the incomplete staining of the microtubules. Hence it is important that the α-tubulin antibody be stored under the manufacturer recommended storage conditions and used within one year.
Using this protocol, high quality multi-color STED images that show the organization and structure of the actin cytoskeleton and the microtubule network, as well as proteins such as IQGAP1 and CLIP-170 that associate with these two cytoskeletons17, could be acquired. The STED images in Figure 4 show the peripheral ring of dendritic actin, as well as microtubules that emanate from a central location in the cell where the actin staining is much less dense. CLIP-170-GFP at the ends of these microtubules is closely associated with the peripheral F-actin. This protocol allows one to visualize cytoskeletal structures at the immune synapse using either single-color STED imaging (Figure 2) or multicolor STED imaging (Figure 3 and Figure 4). However, it should be noted that single color STED imaging (Figure 2) may yield better resolution of actin structures and microtubules in B cells than multi-color STED (Figure 4). This may be due to photobleaching caused by sequential STED image acquisition for the different fluorophores. To obtain the best super-resolution images, the combination of fluorophores and fluorescent proteins selected, as well as the sequence in which they are imaged using the excitation and depletion lasers, should be optimized for the sample. Nevertheless, multi-color STED imaging provides higher resolution images of these cytoskeletal structures than conventional confocal microscopy. Additionally, single-color STED can be used to acquire 3-dimensional super-resolution images of the entire B cell actin or microtubule network (Movie 1).
When using cells transfected with fluorescent fusion proteins, achieving optimal expression levels and avoiding artifacts due to overexpression are significant considerations. In cells where CLIP-170-GFP is overexpressed, large aggregates of CLIP-170-GFP are formed (Figure 5A). In addition to this mislocalization of CLIP-170-GFP, only part of the microtubule network in this cell was within the focal plane closest to the coverslip (Figure 5B). This suggests that CLIP-170 overexpression may also impair BCR-induced MTOC polarization. Conversely, because strong fluorescence signals are typically required for acquiring high quality STED images, low expression of fluorescent fusion proteins such as CLIP-170-GFP (Figure 5C) results in poor quality images. Hence, when using cells that have been transfected with fluorescent proteins, it is important to image only those cells with optimal levels of fusion protein expression. It is also important to note that the transfection protocol that was used for A20 cells (see Table of Materials) typically results in 20-50% of the cells expressing the transfected protein. For plasmid DNA (as opposed to siRNAs), transfection frequencies for primary B cells are often much lower than for A20 cells, requiring the use of B cell lines. Nevertheless, high quality STED images of cytoskeletal elements in untransfected primary B cells can be obtained using this protocol (Figure 6).
Figure 1: Comparison of confocal and STED imaging of F-actin. Confocal images (top) and STED images (bottom) of an A20 cell that had spread on anti-IgG-coated coverslips for 15 min before being stained with Alexa Fluor 532-conjugated phalloidin. Using the confocal STED microscope, the same cell was imaged first by confocal microscopy and then by STED. The initial confocal and STED images are shown, along with the same confocal and STED images after deconvolution. Scale bar: 5 µm. Please click here to view a larger version of this figure.
Figure 2: The actin cytoskeleton at the antigen contact site. A20 cells that had been allowed to spread on anti-IgG-coated coverslips for 15 min were stained with Alexa Fluor 568-conjugated phalloidin and imaged by STED microscopy. The initial STED images were deconvolved. Panels A-B and panels C-E show representative images of two different cells. Panel E shows a 3X enlargement of the region in the white box in panel C. Scale bar for panels A-D: 5 µm. Scale bar for panel E: 1 µm. Please click here to view a larger version of this figure.
Figure 3: STED images of the microtubule network and CLIP-170-GFP. A20 cells expressing CLIP-170-GFP were allowed to spread on anti-IgG-coated coverslips for 15 min before being fixed and immunostained with an α-tubulin antibody plus an Alexa Fluor 532-conjugated secondary antibody. (A) Representative image showing immunostaining of the microtubule network, with CLIP-170-GFP located mainly at the plus-ends of microtubules. (B) Sub-optimal staining and resolution of microtubules due to the use of an outdated stock of α-tubulin antibody. Scale bars: 5 µm. Please click here to view a larger version of this figure.
Figure 4: STED images of the actin and microtubule cytoskeletons. A20 cells expressing CLIP-170-GFP were allowed to spread on anti-IgG-coated coverslips for 15 min before being fixed and stained with Alexa Fluor 568-conjugated phalloidin to visualize F-actin, and with an α-tubulin antibody plus an Alexa Fluor 532-conjugated secondary antibody to visualize microtubules. Panels A-D and panels E-F show representative images of two different cells. Panel D is a 3.5X enlargement of the region in the white box in panel C. Scale bars: 5 µm for panels A-C and E-F; 1 µm for panel D. The image in panel A is the same image used in Figure 3A but includes the overlay of the F-actin channel. Please click here to view a larger version of this figure.
Figure 5: Examples of poor quality STED images due to overexpression or insufficient expression of the GFP fusion protein. A20 cells expressing CLIP-170-GFP were allowed to spread on anti-IgG-coated coverslips for 15 min before being fixed and stained as in Figure 4. (A, B) CLIP-170-GFP overexpression results in large, abnormal aggregates of CLIP-170-GFP (A) and impaired MTOC polarization towards the antigen contact site (B). (C) Compensating for insufficient expression of CLIP-170-GFP by increasing the laser power results in poor quality STED images. Scale bars: 5 µm. Please click here to view a larger version of this figure.
Figure 6: STED image of the microtubule cytoskeleton in primary B cells. Primary splenic B cells were cultured for 6 h with 5 ng/µL BAFF plus 2.5 µg/mL LPS, and then allowed to spread for 15 min on coverslips that had been coated with anti-IgM antibodies. The cells were then fixed and stained with an α-tubulin antibody plus an Alexa Fluor 568-conjugated secondary antibody to visualize microtubules. A representative image is shown. Scale bar: 5 µm. Please click here to view a larger version of this figure.
Movie 1: 3D reconstruction of the B cell microtubule network. A20 cells were allowed to spread on anti-IgG-coated coverslips for 15 min before being fixed and immunostained with an α-tubulin antibody plus an Alexa Fluor 488-conjugated secondary antibody. Z-slices were captured at 0.2 µm step sizes for a total of 37 frames. 3D reconstruction was done using the STED microscope's imaging software. Please click here to view this video. (Right-click to download.)
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 "blurred" fluorescence signal. This protocol describes methods for using STED to image the actin and microtubule cytoskeletons, as well as cytoskeleton-associated proteins.
B cell activation induces remodeling of both the actin cytoskeleton and the microtubule network, with the coordinated regulation of the two cytoskeletons being important for immune synapse formation17,42. The method we present has been optimized for simultaneously imaging the actin and microtubule cytoskeletons at the antigen contact site using multi-color STED, but is equally applicable for single-color STED. Previous studies we conducted on the B cell cytoskeleton highlight that STED can provide new insights into how cellular structures are organized and how they interact with each other. Using confocal and total internal fluorescence (TIRF) microscopy, we had observed that microtubules contact the ring of F-actin at the periphery of the antigen contact site17. Using STED microscopy, we were able to show that the plus ends of microtubules that were marked by the +TIP CLIP-170 associate closely with the dendritic actin network at the cell periphery (see Figure 4).
Multiple factors influence which imaging technique is most suitable for one's specific application. These include the resolution that is required, the structures to be imaged, the labeling technique and its signal-to-noise ratio (i.e., contrast), acquisition time, ease of sample preparation, and reproducibility. Sample preparation for STED is not significantly different than for confocal microscopy, and it combines high resolution with rapid image acquisition. A major advantage of STED is that it is an optical process in which the image is directly acquired from the sample and the resolution can be adjusted by changing the power of the STED laser24. Unlike ground state depletion super-resolution microscopy, which reconstructs images from thousands of successive image captures, extensive computational processing is not required for STED, and the introduction of image reconstruction artifacts is avoided24. However, the contrast in STED images is often low24, in which case post-image processing using software such as ImageJ may be needed to enhance the contrast. This is particularly important for images with dense structures, such as a dendritic actin network. To improve image contrast during image acquisition, one can reduce the depletion laser power and/or apply line or frame averaging. Time-gated STED, which captures photons after a user-set time delay, can increase the resolution by decreasing the area from which photons are collected24,43. We recommend optimizing the STED imaging of cytoskeletal structures at the immune synapse by using a combination of these methods to improve contrast and resolution.
Currently, not all fluorophores are optimal for imaging with STED, and not all fluorophore combinations are suitable for obtaining multi-color STED images. Careful adjustment of the detection ranges is important for ensuring minimal bleed-through of fluorophores into adjacent channels. The combination of fluorophores used in this protocol (i.e., GFP, Alexa Fluor 532, and Alexa Fluor 568) is optimal for multicolor STED super-resolution imaging. Compared to structured illumination microscopy (SIM) and single-molecule localization methods (SMLM), such as photo-activated localization microscopy (PALM), STED is not typically ideal for multi-color imaging. However, we show here that slight over-saturation of fluorophore detection, paired with simple image processing tools, can deliver high-resolution multi-color images of the actin and microtubule cytoskeletons.
This protocol for STED imaging of cytoskeletal structures has revealed new details of cytoskeletal architecture at the B cell immune synapse. Although we optimized this protocol for imaging the actin and microtubule cytoskeletons at the antigen-contact site in B cells, these methods should be applicable to other cell types, especially immune cells (T cells, NK cells, mast cells, etc.) that form immune synapses. Moreover, the utility of this protocol could be extended to coating the coverslips with other ligands or adhesive substrates. However, it is important to optimize the protocol and the image acquisition settings for the cell type and the experimental set-up.
The authors have nothing to disclose.
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.
Cell culture | |||
A20 mouse B-lymphoma cells | ATCC | TIB-208 | Murine B-cell lymphoma of Balb/c origin that expresses an IgG-containing BCR on its surface |
RPMI-1640 | Thermo Fisher Scientific | R0883 | |
Fetal bovine serum | Thermo Fisher Scientific | 12483020 | Heat inactivate at 56 oC for 30 min |
2-mercaptoethanol | Millipore Sigma | M3148 | |
Glutamine | Millipore Sigma | G5763 | |
Sodium pyruvate | Millipore Sigma | P5280 | |
Penicillin-Streptomycin | Thermo Fisher Scientific | 15140122 | Liquid, 10,000 units |
Additional materials for primary B cells | |||
70-µm cell strainer | Corning | 352350 | |
Magnetic bead-based B cell isolation kit | Stemcell Technologies | 19854 | EasySep Mouse B cell Isolation kit |
Lipopolysaccharide (LPS) | Millipore Sigma | L4391 | LPS from E. coli 0111:B4 |
B cell-activating factor (BAFF) | R&D Systems | 2106-BF-010 | |
Name | Company | Catalog Number | Comments |
Transfection | |||
Plasmid encoding CLIP-170-GFP | Gift from Dr. Kozo Kaibuchi (Nagoya Univ., Nagoya, Japan); described in Fukata et al. Cell 109:873-885, 2002 | ||
Recommended transfection reagents and equipment | |||
Amaxa Nucleofection kit V | Lonza | VCA-1003 | Follow the manufacturer's directions for mixing the transfection reagents with the DNA |
Amaxa Nucleofector model 2b | Lonza | AAB-1001 | Program L-013 used |
Falcon 6-well plates, TC treated, sterile | Corning | 353046 | |
Name | Company | Catalog Number | Comments |
Coating coverslips | |||
18-mm diameter round #1.5 cover glasses | Thomas Scientific | 1217N81 | Similar product: Marienfield-Superior catalogue #117580 |
Forceps | Fisher Scientific | 1381242 | Dissecting extra fine splinter forceps |
Falcon 12-well sterile tissue polystyrene tissue culture plate | Corning | 353043 | |
100% methanol | Fisher Scientific | A412-4 | |
Sterile phosphate buffered saline (PBS) without calcium or magnesium | Thermo Fisher Scientific | 10010049 | |
Goat-anti-mouse IgG antibody | Jackson ImmunoResearch | 115-005-008 | For A20 cells |
Goat-anti-mouse IgM antibody | Jackson ImmunoResearch | 115-005-020 | For primary mouse B cells |
Name | Company | Catalog Number | Comments |
Staining | |||
Paraformaldehyde (16% stock solution) | Electron Microscopy Sciences | 15710 | Dilute with PBS to working concentration |
Glutaraldehyde (50% stock solution) | Millipore Sigma | 340855 | Dilute with PBS to working concentration |
Triton X-100 | Fisher Scientific | BP151-500 | |
Saponin | Millipore Sigma | S2149 | |
Bovine serum albumin (BSA), Fraction V | Millipore Sigma | 10735094001 | |
Rabbit anti-tubulin antibody | Abcam | ab52866 | 1:250 dilution recommended but should be optimized |
Alexa Fluor 532-conjugated goat anti-rabbit IgG | Thermo Fisher Scientific | A11009 | 1:100 dilution recommended but should be optimized |
Alexa Fluor 568-conjugated phalloidin | Thermo Fisher Scientific | A12380 | 1:100 dilution recommneded but should be optimized |
Prolong Diamond Antifade Mountant | Thermo Fisher Scientific | P36970 | Without DAPI |
Fisherbrand Superfrost Plus Microscope Slides | Fisher Scientific | 12-550-15 | |
Name | Company | Catalog Number | Comments |
Materials | |||
Parafilm | VWR | P1150-2 | |
HEPES | Millipore Sigma | H3375 | |
NaCl | Fisher Scientific | BP358 | |
KCl | Millipore Sigma | P9333 | |
CaCl2 | Millipore Sigma | C1016 | |
Na2HPO4 | Fisher Scientific | S374-500 | |
MgSO4 | Fisher Scientific | M63 | |
Dextrose | Fisher Scientific | D16-500 | |
Name | Company | Catalog Number | Comments |
Microscopy | |||
Leica SP8 TCS STED microscope | Leica | ||
Huygens deconvolution software | Scientific Voume Imaging | See https://svi.nl/HuygensProducts |