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

Abstract

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.

Figure 1

Immunogold electron microscopy technique. (a,b) 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. 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.

Figure 2

Flowchart showing the main stages and timing of pre-embedding immunogold EM procedure.

Figure 3

Pre-embedding immunonanogold electron microscopy procedures. (a,b) Frozen cells or tissue are cut on a cryostat (10 μm-thick sections) and mounted on glass slides. (c) Slides are immersed in a sequence of blocking solutions using a Coplin staining jar. (d,e) Sections are incubated with the primary antibody, and then they are washed and incubated with the secondary antibody. (f) After a silver enhancement step, sections are processed for conventional electron microscopy. (g,h) Resin embedding is performed by inverting labeled, resin-filled plastic capsules over the slide-attached cell sections. (i) The resulting blocks, detached from the slides, show embedded sections at their surfaces. (j–l) Thin sections, cut on an ultramicrotome, are mounted on grids, stained with lead citrate (Pb) and analyzed on a transmission electron microscope (TEM).

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.. 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; LB, lipid body. Scale bar, 1.0 μm.

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.. 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, 1.5 μm.

Figure 6

Human leukocyte subcellular sites immunolabeled for different antigens using pre-embedding immunoEM. (a) Large vesiculotubular compartments (arrowheads) are positive for major basic protein (MBP), whereas (b) the nuclear envelope (arrowheads) is clearly labeled for protein disulfide isomerase (PDI). Primary antibodies were monoclonal mouse anti-human MBP (a) and monoclonal mouse anti-human PDI (b), as described in Table 1. Secondary antibody was goat anti-rabbit 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. 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 bars, 300 nm (a) and 1.1 μm (b).

Figure 7

Example of immunogold labeling of a mouse tissue (skin). Platelets with specific labeling at the surface of their α-granules (Gr) for nitric oxide synthase 3 (NOS3) are seen in the lumen of a blood vessel. Primary antibody was polyclonal rabbit anti-NOS3, as described in Table 1. Secondary antibody was goat anti-rabbit Fab fragment conjugated to 1.4-nm gold particles (1:100, Nanogold, Nanoprobes, cat. no. 2004). Experimental protocols were approved by the Institutional Animal Care and Use Committee of the Beth Israel Deaconess Medical Center. Scale bar, 500 nm.