[Methods in Cell Biology] Correlative Light and Electron MIcroscopy Volume 111 || Labeling of...

75METHODS IN CELL BIOLOGY, VOL 111Copyright 2012, Elsevier Inc. All rights reserved.

0091-679Xhttp://dx.doi.org/10.1016/B978-0-12-416026-2.00005-4

CHAPTER 5

Labeling of Ultrathin Resin Sections for Correlative Light and Electron Microscopy

Gunar Fabig*,§, Susanne Kretschmar*, Susanne Weiche†, Dominic Eberle*, Marius Ader* and Thomas Kurth*

* Center for Regenerative Therapies, TU Dresden, Fetscherstraße 105, D-01307 Dresden, Saxony, Germany

† Biotechnology Center, TU Dresden, Tatzberg 47-49, D-01307 Dresden, Saxony, Germany

§ Present Address: Medical Theoretical Center, Medical Faculty Carl Gustav Carus, TU Dresden, Fiedlerstraße 42, D-01307 Dresden, Saxony, Germany

Abstract

Correlative microscopy combines the versatility of the light microscope with the excellent spatial resolution of the electron microscope. Here, we describe fast and simple methods for correlative immunofluorescence and immunogold labeling on the very same ultrathin section. The protocols are demonstrated on sections of tissue samples embedded in the methacrylate Lowicryl K4M. Ultrathin sections are mounted on electron microscopy (EM) grids and stained simultaneously with fluorescent and gold markers. For the detection of primary antibodies, we applied either protein A gold or immunoglobulin G (IgG) gold in combination with secondary antibodies coupled

Abstract I. Introduction II. Rationale III. Methods

A. Fixation and PLT-Embedding of Samples for On-Section CLEM B. General Labeling Protocol C. CLEM of GFP-Labeled Rhodopsin in Retinas of Mice Using Prot A Gold and

Fluorochrome-Conjugated Antibodies D. CLEM of α-Tubulin in the Microtubular Manchette of Mouse Spermatids

IV. Materials V. Summary and Outlook

AcknowledgmentsReferences

76 Gunar Fabig et al.

to Alexa488 or Alexa555. Alternatively, the correlative marker FluoroNanogold was used, followed by silver enhancement. The samples have to be analyzed first at the light microscope and then in the transmission electron microscope (TEM), because the fluorescence is bleached by the electron beam. Labeled structures selected at the fluorescence microscope can be identified in the TEM and analyzed at high resolution. This way, fluorescent signals can be directly correlated to the corresponding subcellular structures in the area of interest.

I. Introduction

Light microscopy in combination with a plethora of different fluorescent markers for the analysis of living or fixed cells has revolutionized biology and biochemistry. Recent developments in fluorescence microscopy (FLM) even narrowed the resolution gap between light and electron microscopy (EM) (for recent reviews see, Hell, 2007, 2009). Although conventional FLMs, such as widefield FLM or confocal laser scanning microscopes (CLSM), are limited by diffraction, the super-resolution microscopes, such as stimulated emission depletion (STED) or photoactivatable localization microscopes (PALM), have extended the diffraction barrier down to 20–60 nm (Hell, 2007, 2009). The major drawback of these super-resolution techniques is that they are confined to fluorescence. In other words, one can see the fluorescence emitted from labeled struc-tures but not the unlabeled reference space. EM, on the other hand, is able to reveal those subcellular details at high resolution but cannot be used for in vivo imaging.

Correlative light and electron microscopy (CLEM) combines the best of both worlds, i.e., the versatility of fluorescent markers and the high spatial resolution of the EM, to analyze dynamic subcellular architecture at high resolution (Brown et al., 2009; Gaietta et al., 2002, 2006; Giepmans et al., 2006; Giepmans, 2008; Grabenbauer et al., 2005; McDonald, 2009; Polishchuk et al., 2000; van Rijnsoever, et al., 2008; van Weering et al., 2010; Verkade, 2008). However, many CLEM approaches, in particular, those combin-ing in vivo fluorescence and transmission electron microscope (TEM)-imaging, are used preferentially in cell culture systems (Brown et al., 2009; McDonald, 2009; Polishchuk et al., 2000). CLEM methods are not easily applicable to “large” multicellular specimens, such as tissue samples or whole embryos, with the exception of rather small organisms, such as Caenorhabditis elegans and others (Kolotuev et al., 2010; Müller-Reichert et al., 2008; Sims & Hardin, 2007). Finally, many CLEM approaches focus on single subcel-lular events such as vesicle fusion or budding, and therefore accumulation of data that are sufficient for statistical evaluation is laborious and time consuming.

An alternative approach for the analysis of cells and tissues is the correlative on-section labeling of either resin or Tokuyasu cryosections using fluorochrome- coupled antibodies and gold probes (Cortese et al., 2009; Robinson & Takizawa, 2009; Schwarz & Humbel, 2007, 2009; Takizawa & Robinson, 2006; Vicidomini et al., 2008, 2010). This approach has several of the following advantages: (1) Using sec-tions through cell pellets or tissues, many areas of interest can be analyzed and statisti-cally evaluated. This reduces the risk to lose areas of interest during EM-preparation.

775. Labeling of Ultrathin Resin Sections for Correlative Light and Electron Microscopy

(2) Semithin (200–400 nm) and ultrathin (50–100 nm) sections can be analyzed by FLM. In such samples, fluorescence is emitted from a very thin optical plane, resulting in a very precise signal that is perfectly in focus (Schwarz & Humbel, 2007, 2009). (3) The method is relatively easy to use and widely applicable. (4) Many different antigens may be analyzed on sections from a single sample.

Here, we describe fast and reliable protocols for the correlative staining of ultrathin resin sections for FLM and TEM. Correlative labeling of sections is always performed on sections from the same block/sample. However, fluorescence and gold labeling may be done either separately on adjacent sections (Hoffmann & Schwarz, 1996; Kurth, 2003; Kurth et al., 1996, 1999, 2010; Robinson & Takizawa, 2009; Schwarz et al., 1993a; Schwarz, 1994, 1998; Schwarz & Humbel, 2007, 2009; Takizawa & Robinson, 2006), or on the very same section mounted on a finder grid (Cortese et al., 2009; Vicidomini et al., 2008, 2010). The choice of either method is dependent on the size and abundance of the structures of interest. Here, we focus on the correlative label-ing of single ultrathin sections from resin-embedded tissue blocks. The protocols are demonstrated with two different antibodies and in two different mouse tissues: anti-GFP staining of cells expressing a rhodopsin-GFP fusion protein in the retina, and anti-α-tubulin staining in the spermatids of the testis.

II. Rationale

CLEM, especially the combination of in vivo fluorescence imaging and EM, is a powerful tool to unravel subcellular processes, such as Golgi-dynamics or vesicle traf-ficking, in hitherto unprecedented detail. However, many CLEM approaches focus on cell culture models, and much workload has to be invested to study a single subcellular event. To address the distribution of labeled cells and structures by CLEM in tissues or in embryos, alternative methods are needed such as correlative on-section labeling of semi- to ultrathin sections for fluorescence and EM. Here we provide some simple and reliable protocols for the correlative fluorescence and gold labeling of antigens on single ultrathin methacrylate sections (Lowicryl K4M) mounted on grids. Labeled sections are consecutively analyzed in the FLM and the TEM. This way, many cells or labeled subcellular structures can be analyzed in situ on sections from a single tissue sample.

III. Methods

A. Fixation and PLT-Embedding of Samples for On-Section CLEM

Progressive lowering of temperature (PLT)-embedding into acrylic resins for immu-nocytochemistry has been introduced decades ago to gradually decrease the tempera-ture while the sample is dehydrated in a graded series of ethanol (Carlemalm et al., 1982; for a recent review see Cavalier & Spehner, 2009). Compared to room tempera-ture embedding, PLT reduces the denaturation of proteins and the extraction of lipids


during dehydration and infiltration. At low temperatures, the samples are infiltrated with either Lowicryl K4M (−35°C) or Lowicryl HM20 (−35°C to −50°C) and polymerized by UV-light. PLT-embedding is a versatile embedding method for immuno-EM and a valuable alternative to freeze-substitution (FS) followed by low temperature embedding (Humbel, 2009; Humbel & Schwarz, 1989; Schwarz, Hohenberg, & Humbel, 1993b) or the Tokuyasu method (Slot & Geuze, 2007; Tokuyasu, 1980).

Fixation: For immunolabeling, samples have to be fixed mildly to preserve as much antigenicity as possible (Griffiths, 1993). Commonly used fixatives are 2–4% paraformaldehyde (PFA) with (or without) low concentrations of glutaraldehyde (GA, 0.05–0.5%). The retina samples presented here were fixed by perfusion with 4% PFA in 0.1 M phosphate buffer (PB) pH 7.4 (Figs 1–3), and the testis samples were fixed by immersion after dissection of the animal in 4% PFA in 0.1 M PB pH 7.4 (Figs 4–7).

Alternatively, samples may be cryoimmobilized through high-pressure freezing (HPF), followed by FS and low temperature embedding, a combination of methods considered as the “gold standard” for good preservation of both antigenicity and ultra-structure (for further information see Schwarz & Humbel, 2009; Studer et al., 2001, 2008). However, HPF/FS is limited to samples below 200 µm thickness. Large tissue blocks have to be dissected before HPF, for example, by using a biopsy punch (Studer et al., 2008).

Dehydration: Samples are dehydrated in a graded series of ethanol/water mixtures at progressively lower temperatures according to the following regime: • 30%, 50% at 0–4°C for 45 min each, in the fridge or on ice • 70% (and 80%, optional) at −20°C for 1 h (each), in the freezer • 90%, 96%, 2 × 100% at −35°C, 1 h each, in the Leica AFS2 FS device.

Infiltration: Infiltration occurs in the AFS2 at −35°C • 1 part ethanol/1 part K4M for 1 h • 1 part ethanol/2 parts K4M for 1 h • Pure K4M overnight • Pure K4M 1–2 × 3 h.

Embedding and polymerization: Samples can be processed and embedded in 0.5 mL reaction tubes, or transferred for embedding into flat embedding forms as available for the AFS2 (Leica). Samples are polymerized by UV-irradiation for 24–48 h at −35°C (AFS2 with mounted UV-lamp). Samples in closed 0.5 mL reaction tubes are covered with strips of aluminum foil to trigger polymerization through indirect irradiation by UV-light. This way, irregular polymerization can be prevented. After 12–24 h the alu-minum strips can be removed, and the samples are irradiated for another 12–24 h. After complete polymerization of the resin, the samples are heated to room temperature and the K4M blocks are transferred to a fume hood to get rid of the remaining volatile and potentially dangerous Lowicryl vapors. The samples remain in the fume hood until the characteristic Lowicryl “smell” has vanished.

Sectioning: Blocks are mounted for ultramicrotomy, and semithin sections are pro-duced and stained with toluidine blue/borax (Trump, Smuckler, & Benditt, 1961) to select potentially interesting areas at the light microscope. For correlative immunola-beling, ultrathin sections are collected on slot, mesh, or, preferably, finder grids.


B. General Labeling Protocol

Our general strategy is to label primary antibodies with protein A (Prot A) gold or immunoglobulin G (IgG) gold (10 nm), and subsequently fill up unoccupied binding sites with secondary antibodies conjugated to fluorochromes (Alexa488 or Alexa555) (Fig. 1). Prot A is a bacterial protein that binds the Fc-domain of IgG

Fig. 1 Correlative light and electron microscopy (CLEM) on ultrathin resin sections. (A) Schematic diagram of the procedure. After fixation and embedding, tissue sections are mounted on EM grids and stained simultane-ously for fluorescence light microscopy (FLM) and transmission electron microscopy (TEM). Samples are first imaged in the FLM and then in the TEM, section through the mouse retina: outer segments of photoreceptor cells labeled (red); asterisk, landmark that serves as a reference point. (B) Workflow for CLEM on sections using either Prot A gold/IgG gold (top) or FluoroNanogold (bottom). (C) Fluorescence after TEM-imaging. Fluores-cence is still intact on grid bars (arrows) but completely abolished in areas that were inspected in the TEM (white asterisks). OS layer of the outer segments of the photoreceptors as indicated by dashed lines. (See color plate.)


molecules from different species, such as rabbit, pig, guinea pig, dog, and human. On the other hand, it only weakly binds to antibodies from sheep, goat, donkey, rat (with the exception of IgG2C and IgG1), or mouse (with the exception of IgG2A and IgG2B), and it does not bind to chicken antibodies (Langone, 1982). IgG’s can be raised in different species and can be used as specific secondary detection markers for indirect immunolabeling. Gold particles reduce the binding capacities of both

Fig. 2 CLEM of rhodopsin-GFP in resin-embedded mouse retina. (A) Schematic of the labeling approach. The anti-GFP is a rabbit polyclonal antibody (ab 290 from Abcam). (B) Semithin section of the retina with the characteristic layers, toluidine blue/borax staining. (C) Overview of an ultrathin section at the FLM. The arrow indicates the outer segment displayed in D–F. The asterisks in C and D serve as landmarks and indicate identical positions. (D) Low magnification EM micrograph of the receptor cells. (E) The boxed area indicated in D at higher magnification. (F) The outer segment indicated by the arrowheads in C and D at high magni-fication. The membrane discs are densely labeled. (G–H) Second example illustrating the CLEM approach. (G) Labeled outer segments, fluorescence; boxed area indicates the region displayed in H. (H) Low magnifica-tion EM micrograph. The square indicates an area of an outer segment displayed in I. (I) Immunogold labeling of Rho-GFP a high magnification. (See color plate.)


Fig. 3 CLEM of single transplanted cells. GFP-labeled photoreceptor precursor cells were transplanted into the subretinal space of wild-type tissue. After 3 weeks the mouse was perfusion-fixed and the retina dissected and embedded in Lowicryl K4M. Sections were mounted on finder grids and stained with rabbit-anti-GFP (TP401 from Torrey Pines) followed by Prot A gold and goat-anti-rabbit Alexa488. (A) FLM image, the cluster of transplanted cells is visible at the periphery (arrows), some cells are integrated into the tissue (arrowheads); onl, outer nuclear layer. (B) Some of the integrated labeled cells are highlighted (1–3) and a fold in the section (*) serves as a landmark. (C) Low magnification TEM-image, the labeled outer segments are marked (1–3, green dotted line), an unlabeled outer segment is indicated by the blue dotted line, the landmark fold by an asterisk. (D, E) Two of the labeled cells displayed in A–C are shown at higher magnification (1, 2, green dotted lines). The labeling is clearly visible, as well as the typical substructure of the outer segment. An unlabeled outer segment of the host tissue is also displayed (blue dotted line); os, outer segment. (See color plate.)


Prot A and IgG. In general, labeling intensity decreases with the increasing size of the gold particles (Humbel & Biegelmann, 1992; Humbel et al., 1998). Gold probes of 10 nm, therefore, will probably not occupy all the potential binding sites. In addi-tion, the fluorochrome-conjugated antibodies may bind target IgGs at different sites than Prot A, which may leave even more binding sites for the fluorescent markers (Griffiths, 1993).

The labeling is basically done according to the following procedure (all steps to be carried out at room temperature):

1. 2 × 5 min 1% bovine serum albumine (BSA), fraction V in PBS (hydration of sections and blocking of unspecific binding sites)

2. Primary antibody (rabbit-anti-GFP) in 1% BSA/PBS for 1h 3. 4–5 × 2 min PBS 4. Optional: (a) Bridging antibody, e.g., rabbit-anti-mouse IgG; necessary when the

primary antibody is a mouse monoclonal and Prot A gold serves as a colloidal gold marker (b). FluoroNanogold-conjugated antibody; proceed with washing (Step 5) and then go to Step 13

5. Optional: after bridging antibody or FluoroNanogold: 5 × 2 min PBS 6. Prot A gold or IgG gold in 1% BSA/PBS for 30 min to 1h (gold probe) 7. 3× short changes in PBS

Fig. 4 CLEM of ultrathin resin sections through mouse testis using Prot A gold, a bridging antibody (rabbit-anti-mouse) and goat-anti-rabbit Alexa488. (A) Schematic of the labeling procedure. (B) Overview image showing a section through a seminiferous tubule with α-tubulin staining predominantly in the inner part, where the spermatids differentiate. The square indicates the region displayed in C. (C) Labeling of the spermatid man-chettes is clearly visible, the boxed area indicates the two cells displayed in D and E. (D) High magnification fluorescence image of labeled spermatids; nuc, nucleus. (E) Low magnification EM micrograph of the same area. Squares in D and E indicate the region shown in F; ac, acrosome. (F) EM micrograph showing the gold-labeled manchette (ma). (See color plate.)


8. 4 × 2 min PBS 9. 1% GA in PBS for 5 min in the hood (postfixation; this step is optional and is

dependent on how much time the imaging of specific fluorescence will take; fixes the Prot A gold; can be omitted when IgG gold is used)

10. 4–5 × 2 min PBS 11. Secondary antibody conjugated to Alexa488 or Alexa555 (fluorescence marker)

in 1% BSA/PBS for 30 min (from here on samples have to be protected from light)

12. 4–5 × 2 min PBS 13. 5–10 min DAPI (1 µg/mL, counterstaining of nuclei) 14. 5 × 1 min water 15. Mounting of grids for FLM in 50% glycerin/water between a microscope slide

and a coverslip (Vicidomini et al., 2008) 16. Imaging at the FLM. It is mandatory to finish FLM before imaging at the TEM,

because the electron beam will destroy the fluorescence (Fig. 1(C)) 17. 5 × 1 min water 18. Optional: if FluoroNanogold-conjugated antibodies were used, 5 × 1 min addi-

tional washing steps in water, followed by silver enhancement using the R-Gent SE-Silver enhancement kit (Aurion), incubation 30–60 min

Fig. 5 CLEM of ultrathin resin sections using Prot A gold, a bridging antibody (rabbit-anti-mouse) and goat-anti-mouse Alexa488 for labeling. (A) Schematic diagram. (B) FLM image, the spermatid in the red box is selected for EM-imaging. (C) EM micrograph of the spermatid in B; ma, manchette; nuc, nucleus. (D) Higher magnification of the boxed area in C, immunogold labeling is clearly detectable. (See color plate.)


19. Optional: after silver enhancement: 5 × 3 min water 20. Staining with 2–4% uranyl acetate in water, 5–10 min 21. 3 × 1 min water 22. Sections are dried and imaged at the TEM.

In principle, this protocol is the basis for all experiments described here. Variables are the use of bridging antibodies (Figs. 4 and 5(A)), the use of Prot A gold versus IgG gold (Figs. 2–5 versus Fig. 6), and different secondary fluorochrome-coupled antibod-ies (Figs. 2–6). Alternatively, the primary antibodies are detected with FluoroNanogold that is applied at Step 4 and has to be silver enhanced (Step 18) to get recognizable particles at the EM level (Fig. 7). A summary of the different labeling approaches is displayed in Table I.

Fig. 6 Alternative approaches for CLEM on ultrathin sections using IgG gold in combination with Alexa488 conjugated antibodies. (A) Primary antibodies are detected with goat-anti-mouse IgG gold (10 nm) and goat-anti-mouse Alexa488. (B) Primary antibodies are detected with goat-anti-mouse IgG gold (10 nm) and donkey-anti-goat Alexa488. Arrows in (B) indicate gold particles; ma, manchette; nuc, nucleus. The images show FLM and EM images of indicated areas. (See color plate.)


C. CLEM of GFP-Labeled Rhodopsin in Retinas of Mice Using Prot A Gold and Fluorochrome-Conjugated Antibodies

GFP is among the most commonly used markers for cellular proteins (for a recent review see Giepmans et al., 2006). Although designed for in vivo imaging, it can also be used to identify certain proteins or cell types after fixation of cell and tissue samples. It can be visualized either by its inherent fluorescence, which “survives” many fixation protocols including PFA and moderate amounts of GA (Chalfie et al., 1994; Luby-Phelps et al., 2003), or by immunolabeling with anti-GFP antibodies (Luby-Phelps et al., 2003).

To visualize a translational rhodopsin-GFP (Rho-GFP) fusion protein in the retina of transgenic Rho-GFP mice (Chan et al., 2004), the animals were perfusion-fixed (see Section III A). The eyes were excised and retina pieces, without the removed retinal pigment epithelium (RPE), were embedded in Lowicryl K4M. Ultrathin sections were subsequently mounted on mesh grids and labeled with anti-GFP (rabbit-anti-GFP, ab 290 from Abcam or TP401 from Torrey Pines). Although the GFP-fluorescence can be seen in thin Lowicryl sections and even better in Tokuyasu cryosections (data not shown), the fluorescent signal achieved by antibody staining is much stronger, espe-cially when sections come from older K4M blocks.

Fig. 7 CLEM of ultrathin resin sections using FluoroNanogold. (A) Schematic diagram. (B) FLM image, the square indicates the region shown in C. (C) Staining in the microtubular manchettes of spermatids; the box indicates the cell displayed in D and E at higher magnification (D) Close-up of the cell highlighted in C; nuc, nucleus. (E) Low magnification EM micrograph of the very same cell; squares in D and E indicate the region displayed in F. (F) TEM-micrograph at high magnification with silver-enhanced gold particles in the spermatid manchette (ma). (See color plate.)


Ultrathin sections were mounted on either mesh or finder grids and stained accord-ing to the aforementioned protocol (primary antibodies from rabbit, Prot A gold, and goat-anti-rabbit conjugated to Alexa-fluorochromes as detection markers, see Fig. 2(A)). Two different samples were analyzed (Figs 2 and 3), which are as follows:

First, retinas from Rho-GFP+/− heterozygous mice displayed strong fluorescent stain-ing of the outer segments of photoreceptor cells (Fig. 2(C), and (G)). Selected pieces of outer segments can be imaged in the TEM at higher magnification (Fig. 2(C)–(F) and (G)–(I)). Strong gold labeling in the membrane stacks of the outer segments indicates the Rho-GFP fusion protein (Fig. 2(F) and (I)). Thus, the protein of interest is highly abundant in the sample and could be localized in the clearly defined outer segments of the photoreceptor cells.

Second, a group of photoreceptor precursor cells expressing Rho-GFP were iso-lated from postnatal day 4 transgenic mouse retinas and transplanted into the subretinal space of a wild-type mouse. Most of the transplanted cells form a cluster between retina and RPE, but some GFP-labeled cells seemingly integrate into the photore-ceptor layer (Eberle et al., 2011). After 3 weeks, the mouse was fixed and the ret-ina dissected. Small tissue pieces containing dispersed GFP-positive cells were selected, embedded in K4M, sectioned, mounted on finder grids, and stained (Fig. 3). In addition to the cell cluster on top of the outer segment layer (Fig. 3(A), arrows), single GFP-positive cells can be identified in the photoreceptor cell layer (Fig. 3(A) and (B), arrowheads). At the EM level, these structures were identified as outer segments of photoreceptor cells, labeled with Prot A gold (Fig. 3(C) and (E)). Adjacent outer segments of the host tissue were unlabeled (Fig. 3(E)). This example illustrates that the method is quite effective in identifying a minor cell population within a tissue sample.

Table ISummary of different approaches for correlative fluorescence and gold labeling on ultrathin resin sections.

Primary Ab IgG gold, bridging Ab, Prot A gold, FluoroNanogold

Prot A gold Fluorescent marker Silver enhancement (SE)

Figure

Anti-GFP (R-IgG) Prot A gold G-anti-R Alexa488 or Alexa555

Figs 2 and 3

Anti-α-tubulin (M-monoclonal)

Bridging Ab (R-anti-M IgG) Prot A gold G-anti-R Alexa488 Fig. 4


Bridging Ab (R-anti-M IgG) Prot A gold G-anti-M Alexa488 Fig. 5


G-anti-M 10 nm gold G-anti-M Alexa488 Fig. 6(A)


G-anti-M 10 nm gold D-anti-G Alexa488 Fig. 6(B)


G-anti-M Alexa488 FluoroNanogold F(a,b) fragments

SE Fig. 7

Abbreviations: Ab, antibody; D, donkey; G, goat; IgG, immunoglobulin G ; M, mouse; Prot A, protein A; R, rabbit.


D. CLEM of α-Tubulin in the Microtubular Manchette of Mouse Spermatids

Spermatozoa are continuously generated from a pool of spermatogonial stem cells. After meiosis, they differentiate into spermatids with a condensed nucleus, a long flagellum, and the acrosome at the tip of the spermatid head. These devel-opmental changes include the formation of a spermatid–manchette complex that is rich in microtubules around the nucleus (Fawcett et al., 1971; Kierszenbaum & Tres, 2004). The complex can be visualized with anti-α-tubulin antibodies (mouse monoclonal DM1A, Sigma-Aldrich) and is presented here as a second proof-of-principle example for correlative on-section labeling of resin sections. Mouse testis was dissected, immersion fixed in 4% PFA/PB, and embedded in K4M. Ultrathin sections were mounted on slot grids for further correlative labeling.

1. On-Section Labeling with Prot A Gold and Fluorochrome-Conjugated AntibodiesProtein A has only weak or no binding affinity to antibodies from sheep, goat, don-

key, rat, chicken, and mouse (Langone, 1982). If mouse monoclonal antibodies are to be used, bridging antibodies (preferably from rabbit or guinea pig) have to be added to the protocol (after Step 3, see Section III B). We use rabbit-anti-mouse IgGs (diluted in 1% BSA, incubation for 30 min, followed by washing in PBS, 5 × 2 min). The bridg-ing antibodies are then again detected with Prot A 10 nm gold and goat-anti-rabbit Alexa488 (Fig. 4(A)). The spermatid manchettes are easily detectable at the light microscope, even at low magnifications (Fig. 4(B)). After selecting an area of interest in the FLM (Fig. 4(B)–(D)), the very same area was identified in the TEM (Fig. 4(E)), and the corresponding immunogold labeling visualized at high magnification (Fig. 4(F)).

In a slightly different approach, the fluorescent marker (goat-anti-mouse Alexa488) is directed against the primary antibody (Fig. 5(A)). Although there is competition between the bridging antibody and the fluorescent marker, the fluorescence is of suf-ficient intensity and specificity (Fig. 5(B)) and Prot A gold labeling is apparently not affected (Fig. 5(C) and (D)). In these experiments, we omitted the postfixation step to avoid fixation of bridging antibodies that are not bound to Prot A gold and to enable the binding of fluorochrome-conjugated antibodies.

2. On-Section Labeling with IgG Gold and Fluorochrome-Conjugated AntibodiesWhen IgG gold is used instead of Prot A gold, the bridging antibody can be omitted,

and the primary antibodies are detected with goat-anti-mouse IgG 10 nm gold and goat-anti-mouse Alexa488 (Fig. 6(A)) or donkey-anti-goat Alexa488 (Fig. 6(B)). Correlative labeling works in both cases. The immunogold labeling of the spermatid manchette is weaker, which may be explained by the lack of the bridging step that also enhances the labeling intensity (Slot & Geuze, 2007). However, in other experiments, we experienced that Prot A gold generally revealed more intense staining than the IgG gold probes.

3. On-section Labeling with FluoroNanogoldFluoroNanogold is a correlative marker (Powell et al., 1997; Robinson et al.,

2000; Takizawa et al., 1998). It is an F(a,b)-fragment conjugated to both an Alexa-fluorochrome and an ultrasmall gold compound (Nanogold, about 1–1.4 nm).


Using this approach, correlative labeling is a one-step reaction, after which fluo-rescent and gold markers are both bound to the very same antigen (Fig. 7(A)). However, Nanogold has to be silver enhanced to be detectable at convenient magni-fications in the TEM (Lah et al., 1990; Stierhof, 2009; Stierhof et al., 1991). For the samples displayed here, the R-Gent SE-kit from Aurion has been used according to the manufacturers’ instructions. Again, the spermatid manchettes can be selected in the FLM and identified in the TEM (Fig. 7(B)–(E)). At high magnification, the silver-enhanced gold compound is clearly visible (Fig. 7(F)). The fluorescence, however, was much weaker compared to any other approach presented here. This may be due to quenching by the gold particles, although it has been previously reported not to be a problem with FluoroNanogold (Powell et al., 1997).

IV. Materials

• Fixatives: For immunocytochemistry: 4% PFA in 0.1 M PB pH 7.4 (PB; 0.2 M stock solution: 19 mL of buffer A (27.6g NaH2PO4·1H2O in 1000mL distilled water) + 81 mL buffer B (35.6g Na2HPO4·2H2O in 1000mL distilled water)), or in 100 mM HEPES pH 7.4 or 4% PFA + 0.1% GA with the same buffer options.

• Embedding media: Lowicryl K4M kit (EMS # 14330), work always under fume hood, because all Lowicryl formulations (K4M, HM20, K11M) are potentially dangerous and allergenic.

• Chemicals: BSA (Merck, # 1.12018), GA (25% in water, EMS # 16220), HEPES (EMS # 16782), PFA (granular, EMS # 19208), uranyl acetate (Polysciences # 21447), toluidine blue/borax solution: 1% toluidine blue (EMS # 22050), 0.5% sodium tetraborate (EMS # 21130).

• Primary antibodies: Rabbit-anti-GFP (ab 290 from Abcam, Cambridge, UK; or TP401 from Torrey Pines, San Diego, USA; both antibodies work nicely on Tokuyasu and K4M sections); mouse monoclonal antibody against α-tubulin (DM1A from Sigma-Aldrich, St. Louis, USA).

• Secondary antibodies and detection systems: We used several Alexa488- and Alexa555-conjugated secondary antibodies for immunofluorescence (all from Invitrogen/Molecular Probes), and rabbit-anti-mouse IgG (Sigma-Aldrich) as a bridging antibody. DAPI (Invitrogen/Molecular Probes) served as a counterstain. For EM, we use protein A conjugated to 10 nm gold particles (Aurion, Wageningen, the Netherlands). Alternatively, goat-anti-mouse IgG 10 nm gold (Aurion) was used. Goat-anti-mouse Alexa488 FluoroNanogold F(a,b)-fragments were pur-chased from Invitrogen (# 24920) and the R-Gent-SE-EM silver enhancement kit from Aurion (# 25521).

• Tools and materials: Slot grids (copper and nickel, Plano, # G 255C and # G 2500Ni), hexagonal mesh grids, 100 mesh (copper and nickel, Plano, # G 2410C and # G 2410Ni), 200 mesh finder grids (nickel, EMS, # G 200F1-Ni), anti- magnetic tweezers (model 5B, EMS # 78 320-5B), diamond knives (Diatome, Biel, Switzerland).


• Instruments: Leica AFS2 FS unit and Leica EM UC6 ultramicrotome (both Leica Microsystems, Vienna, Austria); fluorescence microscope: Keyence Biozero (BZ 8000, Keyence, Osaka, Japan), closed digital and inverted fluorescence micro-scope; electron microscope: FEI Morgagni 268D (FEI, Eindhoven, The Nether-lands), operated at 80 kV.

V. Summary and Outlook

Correlative labeling of ultrathin sections for FLM and TEM is a fast and versatile method to analyze the distribution of proteins in the context of tissue architecture and cellular ultrastructure. In the FLM, large areas of a specimen can be scored and specific sites can be selected for EM, where labeled and unlabeled structures are analyzed simul-taneously at high resolution. Correlative labeling can be successfully performed using fluorescent and gold markers in different combinations (Prot A gold or IgG gold, different fluorochrome-conjugated antibodies, FluoroNanogold). In our laboratory we prefer to use Prot A gold as the colloidal marker, because it is a versatile probe that binds to antibod-ies from several species (see Section III B). If mouse monoclonals are used as primary antibodies, detection occurs with rabbit-anti-mouse bridging antibodies and Prot A gold.

By labeling serial thin sections it may even be possible to search rare cell popula-tions or subcellular structures in larger tissue volumes. The protocol presented here can easily be adapted to an automated approach using an immunogold labeling automat (EM IGL, Leica Microsystems, data not shown). This way, many grids with correla-tively labeled sections can be analyzed in the FLM, and a significantly lower number of “interesting” samples be selected for further EM analysis.

FLM, however, is not only useful to select areas of interest. Semithin to ultrathin sections (50–400 nm) that were prepared for EM are excellent samples for high-res-olution light microscopy (Robinson and Takizawa, 2009; Schwarz & Humbel, 2007, 2009; Vicidomini et al., 2008). First, on-section immunolabeling does not demand the deleterious permeabilization steps with detergents or organic solvents, routinely used for the immunolabeling of cell culture models (Melan & Sluder, 1992; Schnell et al., 2012). Therefore, the preservation of subcellular structures is much better than in rou-tine tissue culture or histology samples, especially when the EM samples are prepared by HPF, FS, and low temperature embedding (Schwarz & Humbel, 2007, 2009).

In addition, there is nearly no out-of-focus signal blurring the image, because the section thickness is below the depth of focus of high aperture objectives resulting in high z-resolution. Therefore, fluorescent samples can be analyzed with nonconfo-cal widefield microscopes. However, it may be promising to combine ultrathin sec-tions with super-resolution microscopes (e.g., 4Pi, STED, PALM, STORM) to further improve the resolution of labeled structures at the light microscopical level. CLEM with 4Pi-imaging of living cells and subsequent TEM-analysis has been recently performed using a cell culture model (Perinetti et al., 2009). However, even at the highest possible resolution using super-resolution FLM, one thing is still missing––the subcellular reference space including the nonlabeled structures in the vicinity of


the fluorochromes or fluorescent proteins. This information can only be obtained from the electron microscope and this is the reason why it is advantageous to combine even super-resolution LM with EM.

Finally, a series of correlatively labeled sections can be used for 3D reconstruction of tissue blocks. This option may be promising for the study of small populations of labeled cells and their interactions with other nonlabeled cells (e.g., adult stem and progenitor cells, transplanted cells (Fig. 3), or embryonic cells, such as neural crest or primordial germ cells). With this approach, larger volumes can be reconstructed. The z-resolution is limited to section thickness, but a thickness of 50–70 nm may be suffi-cient for many studies. Smaller structures can be reconstructed by electron tomography of 200 nm thick CLEM-sections (Cortese et al., 2009; Vicidomini et al., 2008, 2010).

Taken together, on-section CLEM is a rapid and versatile method for the correlative analysis of potentially numerous antigens on sections from the very same cell or tissue sample. Our protocols are based on standard labeling techniques and do not require additional sophisticated instrumentation. To increase throughput, however, the use of immunogold labeling automats, like the Leica EM IGL, or the imaging of labeled sec-tions in an integrated fluorescence and electron microscope as described by Agronskaia et al. (2008) might be useful.

Acknowledgments

The authors wish to thank Katrin Daniel and Attila Toth for mouse testis samples, John Wilson for Rho-GFP mice, and the European Fund for Regional Development (EFRE) for financial support.

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