Pre-embedding immunogold labeling to optimize protein localization at subcellular compartments and membrane microdomains of leukocytes

Precise immunolocalization of proteins within a cell is central to understanding cell processes and functions such as intracellular trafficking and secretion of molecules during immune responses. Here we describe a protocol for ultrastructural detection of proteins in leukocytes. The method uses a pre-embedding approach (immunolabeling before standard processing for transmission electron microscopy (TEM)). This protocol combines several strategies for ultrastructure and antigen preservation, robust blocking of nonspecific binding sites, as well as superior antibody penetration for detecting molecules at subcellular compartments and membrane microdomains. A further advantage of this technique is that electron microscopy (EM) processing is quick. This method has been used to study leukocyte biology, and it has helped demonstrate how activated leukocytes deliver specific cargos. It may also potentially be applied to a variety of different cell types. Excluding the initial time required for sample preparation (15 h) and the final resin polymerization step (16 h), the protocol (immunolabeling and EM procedures) can be completed in 8 h.


Limitations
One limitation of this protocol is that it does not facilitate colocalization studies (i.e., simultaneous observation of two epitopes), which can be achieved with postembedding EM using larger gold probes with different sizes. The protein of interest is investigated by immunolabeling with a primary antibody against the target molecule, followed by a secondary antibody (against the primary antibody) conjugated with gold nanoparticles. In our protocol, we use affinity-purified Fab fragments conjugated with 1.4-nm gold particles (Nanogold). In c, an electron micrograph shows subcellular sites of a human eosinophil leukocyte labeled for CD63. Cell surface microdomains and cytoplasmic secretory granules (Gr) and large vesicles (arrowheads) were labeled. Primary antibody was monoclonal mouse anti-human CD63, as described in table 1. Secondary antibody was goat anti-mouse Fab fragment conjugated to 1.4-nm gold particles (1:100, Nanogold, Nanoprobes, cat. no. 2002). Cells were isolated from the blood of healthy donors as described 23 . Written informed consent was obtained from donors in accordance with the Declaration of Helsinki, and Institutional Review Board approval was obtained from the Beth Israel Deaconess Medical Center. N, nucleus. Scale bar, 700 nm.

Experimental design
This protocol describes a pre-embedding immunogold EM protocol for the detection of proteins in leukocytes. Our methodology, a combination of different strategies for optimal labeling and morphology preservation, has been used for almost 10 years (refs. 17,20-24,27). Figure 2 presents a summary of all the stages of the protocol.
Sample fixation. Because paraformaldehyde has less effect on antigenicity, our experience is that most cells should be first fixed with this fixative alone, labeled and then re-fixed with glutaraldehyde, which is a strong cross-linker of proteins and provides best structural preservation, but is known to lower protein antigenicity 28 . However, a combination of both aldehydes may work for certain antigens. We were not able to detect interleukins within leukocytes even when small amounts of glutaraldehyde (0.1%, vol/vol) were used. However, in our hands, MBP-1 was clearly labeled within human leukocytes by using high concentrations of glutaraldehyde. Therefore, because this protocol can be applied to different cell types and antigens, optimization of the best fixative may be necessary to adapt the protocol to a specific experimental condition. To strive for the best structural preservation, the fixative should be freshly prepared and added directly and gently to the cell suspension; i.e., it is not necessary to centrifuge the cells into a pellet before fixation. Moreover, we do not use a high centrifugation speed (1,000g) for the subsequent step of cell washing with buffer. After fixation, isolated cells are pre-embedded in agar to facilitate cell processing and to prevent mechanically induced artifacts. Our experience is mostly with cells in suspension (such as leukocytes isolated from the peripheral blood or pleural cavity), but we were also successful in labeling antigens in tissue fragments using this methodology. After fixation, samples must be immersed in 30% (wt/vol) sucrose overnight to avoid ice crystal formation during freezing. Storage in liquid nitrogen is preferable to −80 °C because ice crystals can still be formed at this temperature. Our experience is that samples stored in liquid nitrogen can be stored for years and assessed for different antigen immunolocalization studies. This is particularly important for precious human samples.
Immunolabeling. Our approach performs immunolabeling on 10-µm-thick cryostat sections, which enable antibody penetration throughout the section thickness, as previously reported 29 . Therefore, it is not necessary to include a permeabilization step, as required for whole cells 30 . The permeabilization step, which can interfere with the structural integrity of membranes, can be detrimental to the ultrastructure, and thus it is best avoided. Thicker sections are not adequate, as the antibody penetration may be restricted. Sections are collected on special glass slides. Regular uncoated glass slides will not allow adequate adhesion of the cells to the slide surface. Before immunolabeling, nonspecific binding sites need to be blocked. Blocking is particularly important because immunolabeling of leukocytes can result in a strong and unexpected background formation. We use two steps of blocking buffers before incubation with the primary antibody.
Silver enhancement. This stage consists of incubation with a silver enhancement solution according to the manufacturer's instructions, and it enables nucleation of silver ions around gold particles. These ions precipitate as silver metal, and the particles grow in size facilitating observation under TEM. Before silver enhancement, cells are sequentially transferred to distilled water because buffer traces can interfere with the silver enhancement process. Therefore, adequate washing in distilled water must be done before this step. The silver enhancement solution is stored at −20 °C, but it is used at room temperature (20 °C). Therefore, keep in mind that it is necessary to thaw the solution kit before this step. Moreover, the solution is light-sensitive, and the incubation is done in a dark room. We have found that 10 min of incubation is ideal for optimal visualization of the tested antibodies ( Table 1) in leukocytes. However, this time can be changed depending on the antigen. The silver layer covering the gold particles is sensitive to the action of osmium tetroxide, which can induce particle loss. To prevent this, our approach uses a procedure with sodium thiosulfate after the silver enhancement step in order to stabilize the silver-enhanced immunogold particles. Moreover, the time of treatment with osmium tetroxide is just 10 min.
Processing for EM. This stage involves conventional procedures for EM (postfixation with osmium tetroxide, en bloc staining with uranyl acetate, dehydration in alcohol, infiltration and embedding in resin), which is usually time-consuming. Our technique uses a faster infiltration/embedding step compared with most immunoEM procedures, which use an overnight infiltration stage. We have found that 15 min is adequate for the subsequent embedding step.
Ultrathin sectioning. Thin (~80 nm) sections are obtained directly from the block surfaces without the need to cut thick 1-µm sections. Sections are mounted on uncoated 200-mesh copper grids, stained with lead citrate only and viewed with a transmission electron microscope. Bring the mixture to a final volume of 1 liter (10 × dilution) with

(a) For cells in suspension
(i) Carefully add the primary fixative directly on the cell suspension in plastic 10-ml test tubes. For 1-2 ml of cell suspension (for example, obtained after leukocyte isolation from the peripheral blood or cavity lavages), fill the tube to 10 ml with the fixative.  crItIcal step The amount of cells is crucial for obtaining a good visible pellet. In our experience, 3 million cells is the minimum necessary to make one reasonable pellet. However, it is desirable to have more cells and to prepare a more substantial pellet for better visualization during sectioning and processing. Moreover, at least two pellets for each condition should be prepared, if possible, in case something unexpected happens during processing. (ii) Rock the tube gently and fix for 30 min at room temperature. (iii) Remove the fixative by centrifugation at 1,000g for 10 min at room temperature. (iv) Wash the samples in PBS 0.02 M buffer and then resuspend the cells in a small amount of buffer (~500 µl-1 ml) to facilitate subsequent preparation of agar pellets.
? troublesHootInG (b) For tissues (i) Immerse the collected fragments (~1 cm size) in ~5 ml of primary fixative in glass vials.  crItIcal step Tissue must be properly fixed immediately after interruption of blood supply. An interval of 10-15 min could be enough to cause artifacts. Do not ever allow the tissue to dry out. Handle the fragments gently to avoid mechanical damage. (ii) After ~5 min, cut the fragments into small blocks of ~1 mm × 1 mm in size, and then return them to the fixative for additional fixation (60 min) at room temperature. Very dense tissues may require longer fixation (2 h). For cutting, put a small pool of buffer on a piece of dental wax and add the tissue fragment. By using a razor blade, cut the tissue into small pieces. (iii) After fixation, remove the fixative using a pipette and wash the samples in 0.02 M PBS buffer.
2| Refrigerate the samples before further processing.  crItIcal step Because it is generally mildly fixed with 4% (wt/vol) paraformaldehyde, the samples should not be stored at 4 °C for a long time. Paraformaldehyde is not a strong cross-linker of proteins, and fixation can be reversed, damaging the structure. We usually proceed to the next step on the same or next day. 3| Make agar pellets by mixing the agar in 0.02 M (2% wt/vol) PBS and bringing it to a boil on a hot plate until the solution is completely dissolved, clear and just boiling. Leave it on the hot plate, while being stirred with a stir bar, so that the agar stays in a liquid state. Transfer the cell suspension to a microcentrifuge tube and spin it for 1 min (~9,500g) at room temperature. Carefully aspirate the small supernatant from the pelleted cells, quickly add hot agar and resuspend the cells using a toothpick. Spin the cells in a microcentrifuge for 1 min as before.  crItIcal step Cell suspensions must be pre-embedded in agar in order to be cut and processed. This step is not necessary for tissue fragments. In that case, proceed to Step 6.  crItIcal step If the agar is not adequately heated or if melted agar is not quickly added to the microcentrifuge tube, the cells will not re-form a pellet and will be spread out in the solidified agar. ? troublesHootInG 4| Chill the tubes on a bucket of ice for at least 20 min to allow the agar to solidify. After solidification, carefully remove the intact pellets from the microtubes using a razor blade and a pointed applicator stick. Use a small pool of buffer to help handling of the agar blocks and to avoid drying. Cut off any excess agar.  ! cautIon When you are handling liquid nitrogen, gloves, eye protection and closed shoes must be worn.  crItIcal step Freezing of agar pellets containing embedded cell suspensions requires special attention. In our experience, the best way to freeze these pellets is by holding (using a stick) the pellet upright (not horizontally) on the specimen stage. Thus, the area in which the cells are more concentrated will be more visible during sectioning. ? troublesHootInG  pause poInt Store the samples in liquid nitrogen after careful labeling. Frozen samples can be stored for the long term (~3 years).
Immunolabeling • tIMInG ~5 h  crItIcal All steps are performed at room temperature. 7| Cut the frozen sections. Collect 10-µm-thick sections on Superfrost Plus glass slides (Fig. 3a,b). Before sectioning, label each slide with the appropriate code using a pencil (not a permanent pen) and, during sectioning, keep the slide outside the cryostat.  crItIcal step Before collecting all sections that will be immunolabeled, it is important to confirm that the cells and tissues are in adequate numbers and that they are visible on the sections by quickly staining one section with a histological stain. We use a toluidine blue solution that is also used for staining of TEM thick sections in our laboratory. Quickly dry a section on a glass slide by placing the slide on a slide warmer; cover it with a few drops of staining solution, rinse off excess stain gently with distilled water, dry the slide, coverslip with regular mounting medium and analyze it on a light microscope. If sufficient cells are not visible, cut extra sections and repeat this analysis. For immunolabeling, we usually collect four sections per slide for each sample. It is important to collect several sections because some of them may be lost during processing. Each section will correspond to one resin block, and therefore there will be several blocks from each sample by the end of the process. For each antigen to be immunolabeled, it is necessary to have a control for the primary antibody (replaced by an irrelevant antibody), and it is also desirable to have a slide in which the primary and/or the secondary antibody will be omitted. Therefore, for immunolocalization of one antigen in one sample, three slides from the same sample have to be prepared. ? troublesHootInG 8| After sectioning, circle the sections with a diamond scriber pen to facilitate visualization of the section area, and allow the sections to adhere to the slide for ~8 min at room temperature before subsequent steps.
9| Immerse the slides containing the sections in 0.02 M PBS for 5 min. This and other steps requiring slide immersion are performed in Coplin staining jars, as shown in Figure 3c. We use small staining jars that hold a maximum of eight slides. Avoid large jars, which will consume large volumes of solutions. Moreover, our experience is that each experiment should be done with a small number of conditions to facilitate processing.
10| Immerse the slides in PBS-glycine solution for 10 min. Discard PBS and add PBS-glycine solution in the staining jar (Fig. 3c).  crItIcal This step is important for fixative quenching.
11| Block nonspecific binding sites with PBS-gelatin-BSA buffer for 30 min. Discard the PBS-glycine solution and add the first blocking buffer (PBS-gelatin-BSA) to the same jar containing the slides (Fig. 3c).
12| Block nonspecific binding sites with PBS-gelatin-BSA-NGS buffer for 30 min. Discard the first blocking buffer and add PBS-BSA-NGS to the same jar containing the slides (Fig. 3c).
13| Incubate the sections with primary antibody for 1 h. Prepare appropriate dilutions of the primary antibodies immediately before incubation. Antibodies are diluted in PBS-gelatin-BSA-NGS buffer, previously filtered in a coarse filter paper. A list of tested antibodies and their dilutions is shown in table 1. Carefully place slides in a black moisture chamber, and cover each slide with the antibody solution (Fig. 3d,e). Cover the chamber with a black lid.
14| Block the sections with PBS-gelatin-BSA-NGS for 30 min. Discard the antibody solution; place the slides in a staining jar, as shown in Figure 2c, and add PBS-gelatin-BSA-NGS buffer.
15| Incubate the sections with secondary antibody for 1 h. Prepare appropriate dilutions of the secondary antibodies (Nanogold) immediately before incubation. Antibodies are diluted in PBS-gelatin-BSA-NGS buffer, previously filtered in a coarse filter paper. Usually 1:100 dilution works well for our tested antibodies (table 1). Carefully place the slides in a black moisture chamber and cover each slide with the antibody solution (Fig. 3d,e). Cover the chamber with a black lid. 16| Wash the sections in PBS-gelatin-BSA buffer for 3 × 10 min. Discard the antibody solution; place the slides in a clean staining jar and add PBS-gelatin-BSA buffer.
17| Wash the sections in PBS for 3 × 5 min. Discard the PBS-gelatin-BSA buffer and add PBS to the same jar containing the slides.
18| Fix the sections in 2% (vol/vol) glutaraldehyde for 10 min. Move the slides to another clean staining jar and, inside a fume hood, add 2% glutaraldehyde. silver enhancement of nanogold particles • tIMInG ~1 h  crItIcal These steps should be performed at room temperature.
19| Wash the slides in distilled water for 5 × 5 min. Discard the fixative in an appropriate waste container. Place the slides in another clean jar and add distilled water.
20| In a darkroom, mix HQ Silver according to the manufacturer's instructions (Fig. 3f). Place the slides in a clear incubation chamber and cover each slide with the freshly prepared silver solution for 10 min at ~20 °C.  crItIcal step Incubation time affects the growth of gold particles, and therefore time must be rigorously controlled with a timer after covering each slide. ? troublesHootInG 21| Wash the slides in distilled water for 5 × 5 min by immersing them in a clean jar with distilled water.
22| Replace distilled water with 5% (wt/vol) sodium thiosulfate for 3 min. eM processing: postfixation, contrasting and resin embedding • tIMInG ~2 h plus 16 h polymerization  crItIcal All steps are done at room temperature and in a fume hood.
23| Postfix the sections in osmium tetroxide for 10 min. Place the slides in an incubation chamber inside a fume hood. Cover the slides with the osmium solution and cover the chamber with a black lid or aluminum foil.
24| Wash the slides quickly in distilled water.
25| Stain en bloc with uranyl acetate for 5 min. Discard the osmium solution and cover slides with the uranyl acetate solution in the incubation chamber inside a fume hood. Cover with a black lid or aluminum foil.
26| Dehydrate the samples sequentially in 50, 70 and 95% (vol/vol) ethanol for 5 min each, followed by 100% ethanol for 1 × 10 min. Dehydration is performed with the slides in a staining jar by replacing the alcohol series. The jar should be covered after immersing in 100% ethanol.
27| Infiltrate in a mixture of propylene oxide and Eponate (working solution) at a 1:3 ratio for 15 min. This mixture should be prepared in a plastic tri-pour and covered before use. Infiltration is done by placing the slides in a clear incubation chamber and covering the sections with the resin mixture. Do not cover the incubation chamber with a lid.
28| Before embedding, stand the slides up for ~5 min to drain off the excess of infiltration mixture. Use a marker pen to make a circle on the bottom of each section (slide bottom) to facilitate identification of the section area when inverting the capsules. Embed the samples by inverting Eponate-filled plastic capsules over the slide-attached cell sections (Fig. 3g,h).  crItIcal step Labels with the sample codes (written using a pencil or typed) should be inserted inside the capsules before filling them up with resin for appropriate identification. Put the labels on the bottom of the capsules.

29|
Polymerize the samples at 60 °C in an oven for 16 h. ? troublesHootInG 30| Separate the Eponate blocks from the glass slides. Place the slides in a bucket containing a small amount of liquid nitrogen. Cover the bucket with a lid and wait for 1-2 min. Blocks will easily detach from the slides. If small pieces of glass remain on the block surface, immerse the block again in liquid nitrogen for some seconds. Remove the blocks from the plastic capsules by using a BEEM capsule press. Embedded sections will be at the block surface (Fig. 3i). thin sectioning of blocks and eM analysis • tIMInG ~1 h per sample 31| Under a stereomicroscope, localize the area of interest on the block surface (Fig. 3i). Circle this area with a marker pen to facilitate localization, if necessary. By using a new razor blade that has been rinsed with alcohol and dried, trim the block face, to include the area of interest, forming a trapezoid shape ~0.5 mm wide and 0.7 mm high. Ensure that the top and bottom sides of the trimmed block are perfectly parallel, so that the sections will come off the knife in a ribbon.
32| Place the trimmed block in the ultramicrotome (Fig. 3j). Mount the diamond knife (1.8 mm, 45°), fill the water trough with filtered double-distilled water and align the block left to right and top to bottom so that the block face is perfectly parallel to the edge of the knife. Collect ultrathin sections of ~80 nm (silver or gold color) on uncoated 200-mesh copper grids, being careful to avoid folding of the sections. To help pick up thin sections onto each grid, use ultramicrotomy aid tools such as a loop (MATERIALS). Alternatively, a clean eyelash attached to a wooden stick is helpful to guide the sections. Prepare 2-3 grids from each block. Place grids in a small plastic Petri dish with a Whatman filter paper placed underneath the grid (Fig. 3k). Write the block code on the paper (Fig. 3k). Cover it and allow it to dry. ? troublesHootInG 33| Enhance contrast of ultrathin sections (staining) by setting up a Petri dish with dental wax and placing drops of lead citrate on it. Place the grids onto drops (sections side down) for ~3-5 min. Remove the grids individually and rinse thoroughly with double-distilled water (ten dips each). Place the grids on the filter inside the same plastic Petri dish (Fig. 3k). We write the code Pb on the filter paper to distinguish between stained and unstained grids (Fig. 3k). We recommend keeping one unstained grid as reserve. The grids are now ready to be analyzed by TEM (Fig. 3l), or they can be stored in a plastic grid box for subsequent analysis. ? troublesHootInG  pause poInt Grids can be stored at room temperature for several months.  If the initial pellet was made with enough cells, this problem is probably due to inadequate freezing of the pellet (incorrect position). In our experience, pellets should be frozen intact and with the bottom area (in which the cells are more concentrated) upright. This will allow better visualization of the cells during cryostat microtomy and also will guarantee sufficient number of cells to be analyzed on the thin sections. It is preferable to have a small area containing high number of cells than a larger area with spread cells. In this case, you will see a low number of cells on the electron microscope. Re-prepare samples and freeze adequately

20
Excessive growing of gold particles and/or background staining (Fig. 4) The time of incubation with silver enhancement components is not optimized and probably was too long. Room temperature is too high. Silver enhancement is time-dependent. The enhancement time is the time required to obtain an adequate amplification of the Nanogold particles.  Figure 4 | Example of excessive growth of gold particles (arrowheads) and background formation observed in the cytoplasm of a human eosinophil leukocyte after labeling for perilipin 2 (PLIN2/ADRP) and incubation with silver enhancement components for 20 min, which was too long. Primary antibody was polyclonal guinea pig PLIN2/ADRP (reactivity human/mouse/ rat/bovine) (Fitzgerald, cat. no. 20R-AP002). Secondary antibody was goat anti-guinea pig Fab fragment conjugated to 1.4-nm gold (1:100, Nanogold, Nanoprobes, cat. no. 2055). Cells were isolated from the blood of healthy donors, as described in Melo et al. 23 Figure 4 shows excessive growth of gold particles and Figures 5-7 illustrate typical results obtained using this protocol. As shown in Figures 5 and 6 and table 1, human leukocytes can be labeled for different antigens, and gold labels are observed as electron-dense particles associated with subcellular sites. We have used pre-embedding immunogold EM to investigate protein localization at cell surface microdomains (Figs. 1 and 5b; ref. 24), as well as cytoplasmic distribution of varied proteins, such as CD63 (ref . 17; Fig. 5a), MBP 17 (Fig. 6a) and PDI 27 (Fig. 6b), within human leukocytes isolated from the peripheral blood. The technique also provided excellent immunolabeling for tissue antigens from experimental models (Fig. 7) and humans. As a control, cells incubated with an irrelevant primary antibody (Fig. 5c) or not incubated with primary or secondary antibodies (not shown) were imaged. Figures 1 and 5-7 also demonstrate that very good preservation and imaging of cell features, such as the nucleus, nuclear envelope, secretory granules, vesicles, lipid bodies, and endomembranes and plasma membranes, can be expected using this protocol even in suboptimally fixed specimens. Analysis of labeling density in relation to these morphologically identifiable cell organelles and structures can be performed. Values are usually expressed as the number of gold particles per µm 2 (organelle profiles) or per µm in case of membrane traces (reviewed in ref. 31). For example, quantitative analyses of gold nanoparticles using our methodology showed prominent accumulation of PDI on the nuclear envelope (Fig. 6b), with a mean level of labeling of 33.2 ± 9.5 gold particles/µm (mean ± s.e.m., n = 30 cell sections; ref. 27). Moreover, the quality of cell morphology obtained using this protocol is sufficiently adequate to uncover pathological and morphological changes. For example, Figure 5a illustrates the diagnostic appearance of a cell secretory process termed piecemeal degranulation, characterized by emptying of secretory granules in response to an inflammatory stimulus 32 Figure 5 | Examples of pre-embedding immunonanogold labeling of human leukocytes. (a,b) Electron micrographs of eosinophils isolated from the peripheral blood and labeled for CD63 (a) or CD9 (b). Note the dense labeling at secretory granules (a, white arrowheads) and cell surface (b, black arrowheads). In c, a representative cell in which the primary antibody was replaced by an irrelevant antibody shows negative labeling. Primary antibodies were monoclonal mouse anti-human CD63 (a) and monoclonal mouse anti-human CD9 (b), as described in table 1. In c, the irrelevant antibody was an isotype IgG1 control (BD Pharmingen). Secondary antibody was goat anti-mouse Fab fragment conjugated to 1.4-nm gold particles (1:100, Nanogold, Nanoprobes, cat. no. 2002). Cells were isolated from the blood of healthy donors, as described in Melo et al. 23  coMpetInG FInancIal Interests The authors declare no competing financial interests.