Immuno-Electron Microscopy and Electron Microscopic In Situ Hybridization for Visualizing piRNA Biogenesis Bodies in Drosophila Ovaries

Abstract

Immuno-electron microscopy and electron microscopic in situ hybridization are powerful tools to identify the precise subcellular localization of specific proteins and RNAs at the ultramicroscopic level. Here we describe detailed procedures for how to detect the precise location of a specific target labeled with both fluorescence and gold particles. Although they have been developed for the analysis of Drosophila ovarian somatic cells, these techniques are suitable for a wide range of biological applications including human, primate, and rodent analysis.

1 Introduction

PIWI-interacting RNAs (piRNAs) are small noncoding RNAs enriched in animal gonads, where they repress transposons to maintain genome integrity. Loss of piRNAs causes a failure in germline development, resulting in infertility; thus, piRNAs are indispensable for the succession of animal life [1–3].

The majority of piRNAs have sequences antiparallel to transposon transcripts, and so have the potential to act as antisense oligonucleotides to silence them. However, piRNAs do not exhibit any enzymatic activities by themselves. Rather, piRNAs interact specifically with PIWI proteins to form piRNA-induced silencing complexes (piRISCs) and direct them to target transcripts through RNA–RNA base-pairings. Upon this, repression of transposons occurs at either the transcriptional or posttranscriptional levels, depending on the activity and/or the cellular localization of each PIWI protein. A subset of PIWI proteins contain an RNase-H-like endonuclease (Slicer) activity and induces posttranscriptional silencing (target RNA cleavage), whereas other PIWI members lack the nuclease activity, but collaborate with other factors to accomplish transcriptional silencing in the nucleus [4, 5].

Piwi (P-element induced wimpy testes) is one of three PIWI proteins expressed in Drosophila, and was originally identified as a factor necessary for germline stem cell renewal. Later, Piwi was shown to be associated with small RNAs called “repeat-associating small-interfering RNAs (rasiRNAs)” [6–8]. This association of PIWI proteins with rasiRNAs is a biological event conserved among many animal species. Thus, the alternative name “piRNAs” was given to rasiRNAs.

piRNAs originate primarily from intergenic piRNA clusters through the primary processing pathway, and are subsequently amplified by the Ping-Pong cycle, a reciprocal target RNA cleavage event that depends on the Slicer activity of PIWI proteins. Interestingly, the Ping-Pong cycle is germ cell-specific, meaning that somatic cells in the gonads lack the machinery, and therefore, they contain exclusively primary piRNAs. Such cell type-specific bias is also observed for PIWI expression, i.e., among all the PIWI members, Piwi is expressed in the somatic cells, whereas the germ cells express all PIWI proteins [3–5, 9].

The current model for primary piRNA biogenesis in Drosophila ovarian somatic cells (OSCs) shows that perinuclear Yb bodies are the center for processing primary piRNAs and piRISC formation [10, 11]. According to this model, nascent piRNA-free Piwi is localized at Yb bodies through association with Armitage, a DEAD-box RNA helicase. Piwi is then loaded with piRNA intermediates at Yb bodies, which are further processed by unknown factors. This sequential processing gives rise to mature piRISCs, which are then transported to the nucleus, the final destination of the silencing-capable effector complexes.

The notion that Yb bodies are the center for primary piRNA processing was further supported by the observation that many piRNA factors accumulate in these bodies. Depletion of the factors results in a severe primary piRNA loss, although Yb bodies are still present in the cells. However, loss of Yb protein, which consists of a DEAH-box RNA helicase and a Tudor domain, abolishes Yb bodies from the cells. Thus, Yb is the core factor for Yb body formation [10–15].

A recent study by Murota et al. showed that piRNA intermediates arising from the soma-specific piRNA cluster flamenco (flam) accumulate to perinuclear bodies adjacent to Yb bodies in OSCs and follicle cells in the ovaries [16]. Because of this, the non-membranous structures rich in flam-piRNA intermediates were termed Flam bodies. Interestingly, abolition of Yb’s RNA-binding activity by introducing mutations into the DEAD-box helicase domain disrupts not only Flam bodies but also Yb bodies. In parallel, crosslinking immunoprecipitation (CLIP) experiments showed that Yb protein directly associates with flam-piRNA intermediates. Based on these observations, a new model for primary piRNA biogenesis in OSCs was proposed: Yb directs the perinuclear localization of piRNA intermediates and piRNA factors to Flam bodies and Yb bodies, respectively, through direct binding to facilitate piRNA biogenesis and function. This also served to explain the requirement for Yb protein in the piRNA pathway.

In the study, Murota et al. performed RNA fluorescence in situ hybridization (RNA-FISH) to examine the cellular localization of flam-piRNA intermediates, and found that these RNAs accumulate to Flam bodies [16]. However, to obtain further and stronger support, Murota et al. also took an electron microscopy (EM) approach, through which they successfully showed that Flam bodies are bona fide cytoplasmic structures [16]. EM is a widely used method to analyze biological ultrastructure, including molecules, cells, and tissues from a variety of species. In EM, accelerated electrons can visualize subcellular structures with higher resolution than can be obtained by fluorescence microscopy because they have a wavelength shorter than that of visible light. Conventionally, the expression level and the specific location of proteins and RNAs in vivo are evaluated by fluorescence immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH), respectively. Higher-resolution localization of proteins/RNAs can be obtained with immuno-EM (iEM) and EM-ISH [17, 18]. In addition to the high resolution, these approaches can be used to observe the adjacent subcellular organelles or microstructures such as mitochondria, endoplasmic reticulum, ribosomes, myelin lamellae, synapses, cilia, and a variety of molecules and fibers co-localized with the specific proteins or RNAs [16, 19–27].

Here, we describe the methods for how to carry out iEM and EM-ISH, both for cells and for tissues (Fig. 1). Briefly, for general EM analysis, cultured cells and tissues are fixed, dehydrated, embedded into plastic, ultrathin sectioned, and observed with EM (left column in Figs. 1 and 2). Cells and tissues are fixed moderately and the target protein is labeled with gold using a specific antibody to identify specific protein localization by iEM. Then, the samples undergo the next steps including additional EM fixation, a silver enhancement step (second column in Fig. 1) and transfer to the EM procedures (Fig. 3) [19–21, 23–27]. Cells and tissues are fixed and labeled with a tagged probe and a tag-recognizing antibody followed by the same procedures as for iEM (third column in Fig. 1) to visualize specific RNA location by EM-ISH (Fig. 4) [16, 22]. There are two common iEM procedures, designated pre-embedding and post-embedding iEM, which depend on the order of the antibody-staining and the resin-embedding steps (white-colored letters in Fig. 1). In this chapter, we focus on describing pre-embedding iEM.

Fig. 1

Simplified flowchart of electron microscope analyses to identify protein and RNA localization. Sample preparation steps for general EM, pre-embedding iEM, EM-ISH, and post-embedding iEM. The detailed procedures for pre-embedding iEM and EM-ISH are described in the main text. Note that the EM-ISH procedure for tissue samples indicated in this flowchart is specific to mouse samples; however, the description in the text (Subheading 3.4) is specifically for fresh frozen samples including human biopsy or autopsy samples. Human tissue is not fixed initially, meaning that fresh frozen samples immediately after removal from the body should be stocked at −80 °C freezer, used for sectioning, then the EM-ISH procedure starts with fixation in 4 % PFA as indicated in the main text

Fig. 2

Representative results from EM observation. Typical EM images of cultured cells from (a) Drosophila OSCs and (b) S2 cell lines. (c) Typical EM image from mouse brain cortical tissue. N nucleus, arrows myelinated neural fibers, arrowheads synapses. Scale bars are 2 μm

Fig. 3

Images from iEM and IHC experiments with anti-Yb antibodies. (a) The subcellular localization of the Yb protein is clearly shown by gold particles (black dots) in control OSCs. (b) Higher magnification of (a) demonstrates that the gold aggregation (Yb protein localization) is specifically detected adjacent to the mitochondria. (c) Fluorescence IHC enables visualization of a limited number of Yb aggregates in the OSC cytoplasm. (d–f) Yb aggregation was dramatically altered in Zucchini (Zuc)-knockdown OSCs (designated as siZuc), as evaluated with (d) low- and (e) high-magnification iEM; the corresponding fluorescence IHC image is indicated in (f). Detailed interpretation and procedures have been described previously [10, 11, 13, 28]. N nucleus, M mitochondria, DAPI nuclear stain. Scale bars are 2 μm

Fig. 4

EM-ISH and FISH images with a probe against Flam. (a) Subcellular aggregation of Flam noncoding RNAs is indicated by gold particles (black dots) in 0.3 % Triton X-100-treated control OSCs. (b) Higher magnification of (a) reveals a cytoplasmic localization. (c) Fluorescence IHC also shows cytoplasmic aggregation in OSCs. The EM-ISH images from Proteinase K (ProK)-treated OSCs were not clear due to membrane degradation (d, e), whereas the fluorescence IHC results (f) were quite similar to those from the Triton-treated sample (c). N nucleus, DAPI nuclear stain. Scale bars are 2 μm

In our special protocol, it is possible to detect protein/RNA localization with different levels of resolution, both by fluorescence microscopy and by EM [16, 22]. This is what is called correlative microscopic analysis, and it helps to supply additional information to that generated by fluorescence IHC (Figs. 3 and 4). The methods we have developed will enable increased understanding of physiological phenomena and can be applied to many aspects of in vivo biological analysis.

2 Materials

2.1 iEM Analysis of Cultured Cells

• 1. Drosophila ovarian somatic cells (OSCs) [28] (see Note 1).

• 2. Chamber slide (four-well, glass- or plastic-bottomed) (see Note 2).

• 3. Paraformaldehyde (PFA), 16 %, EM grade.

• 4. 10× Phosphate-buffered Saline (PBS): 1.37 M NaCl, 27 mM KCl, 100 mM Na₂HPO₄, 20 mM KH₂PO₄, pH 7.4.

• 5. Saponin: a weak plant-derived detergent.

• 6. 0.1 M Phosphate Buffer (PB).

• 7. Block Ace.

• 8. Mouse anti-Yb monoclonal antibody [16].

• 9. Nanogold- and Alexa Fluor 488-conjugated anti-mouse antibody.

• 10. Glutaraldehyde: 25 % (EM grade).

• 11. HQ-Silver Enhancement kit.

• 12. 4 % osmium tetroxide (OsO₄).

• 13. Ethanol: 50, 70, 80, 90, and 100 % (see Note 3).

• 14. Acetone: 100 %.

• 15. QY-1: 99 % butyl glycidyl ether.

• 16. Epon Plastic Solution: Weigh MNA, EPOK-812, DDSA, and DMP-30 in a cup in the recommended proportions (Table 1) and mixed in a planetary centrifugal mixer for 3.5 min at 2,000 rpm (about 400 × g) followed by deaeration for 1.5 min at 2,200 rpm (about 400 × g) (see Note 4).

  Table 1 Composition of 100 % Epon plastic solution

• 17. Planetary centrifugal mixer.

• 18. Glass slide mold (see Note 5).

• 19. Single-edged razor blade (see Note 6).

• 20. Cut-resistant gloves (see Note 6).

• 21. Diamond knife (2–3 mm width).

• 22. Copper grid (Veco Specimen Grids #100, 150, 200 mesh).

• 23. Grid stick.

• 24. Uranyl acetate: 2 %.

• 25. Lead citrate (Reynold’s Solution).

• 26. Transmission electron microscope (TEM) or scanning electron microscope (SEM) (see Note 7).

2.2 iEM Analysis for Tissue

• 1. Sucrose (powder).

• 2. Frozen section compound.

• 3. Cryomold.

• 4. Liquid nitrogen (N₂).

• 5. Cryostat.

• 6. Matsunami adhesive silane (MAS)-coated glass slides (Matsunami glass, Japan) (see Note 8).

2.3 EM-ISH Analysis for Cells

• 1. PFA: powder.

• 2. 1× PBS, 0.1 M, pH 7.4.

• 3. 50× Denhardt’s Solution.

• 4. Acetylation Buffer: 1.5 % triethanolamine, 0.25 % acetic anhydride, 0.25 % HCl.

• 5. 20× Saline Sodium Citrate Buffer (SSC): 3 M NaCl, 0.3 M C₆H₅Na₃O₇·2H₂O (trisodium citrate dihydrate), pH 7.0.

• 6. Prehybridization Solution: 50 % formamide, 2× SSC, 1× Denhardt’s Solution, 10 mM EDTA, 100 μg/ml yeast tRNA, and 0.01 % Tween 20.

• 7. Hybridization Buffer: 50 % formamide, 2× SSC, 1× Denhardt’s solution, 5 % dextran sulfate, 10 mM EDTA, 100 μg/ml yeast tRNA, and 0.01 % Tween 20.

• 8. Wash Buffer 1: 50 % formamide, 2× SSC, 0.01 % Tween 20.

• 9. NTET: 500 mM NaCl, 10 mM Tris–HCl, pH 8.0, 1 mM EDTA, 0.01 % Tween 20.

• 10. Wash Buffer 2: 2× SSC, 0.01 % Tween 20.

• 11. Wash Buffer 3: 0.2× SSC, 0.01 % Tween 20.

• 12. TBS-T: Tris-buffered saline (TBS) with 0.01 % Tween 20, pH 7.6.

• 13. 10× Blocking Reagent: 0.1 M maleate, 0.15 M NaCl.

• 14. Blocking Buffer: 1× Blocking Reagent in TBS-T.

• 15. 10× Fluorescein RNA Labeling Mix.

• 16. Rabbit anti-FITC (fluorescein isothiocyanate) antibody.

• 17. Nanogold- and fluorescence-conjugated anti-rabbit antibody.

2.4 EM-ISH Analysis for Tissue

• 1. MAS- or poly-l-lysine (PLL)-coated glass slides (see Note 8).

• 2. 0.2 N HCl.

• 3. Proteinase K (PCR grade).

• 4. Acetylation Solution: 1.5 % triethanolamine, 0.25 % acetic anhydride, 0.25 % HCl.

• 5. Prehybridization Solution: 50 % formamide, 2× SSC, 1× Denhardt’s Solution, 10 mM EDTA, 100 μg/ml yeast tRNA, and 0.01 % Tween 20.

• 6. Hybridization Buffer: 50 % formamide, 2× SSC, 1× Denhardt’s Solution, 5 % dextran sulfate, 10 mM EDTA, and 0.01 % Tween 20.

• 7. Wash Buffer 1: 50 % formamide, 2× SSC, and 0.01 % Tween 20.

• 8. RNase Solution: 10 μg/ml RNase A added to NTET.

• 9. Wash Buffer 2: 2× SSC with 0.01 % Tween 20.

• 10. Wash Buffer 3: 0.2× SSC with 0.01 % Tween 20.

• 11. TBS, pH 7.6.

• 12. Blocking Buffer: Blocking Reagent, 0.1 M maleate, 0.15 M NaCl, and 0.01 % Tween 20 in TBS.

• 13. DIG (digoxigenin)- or FITC-labeled probes.

• 14. Primary antibodies against DIG, FITC, and other specific targets.

• 15. Nanogold- and fluorescence-conjugated anti-rabbit antibody.

3 Methods

3.1 iEM Analysis for Culture Cells

The representative images with this iEM procedure are shown in Fig. 3. All steps are performed at room temperature unless otherwise indicated.

• 1. Culture the cells (Drosophila OSCs) in a slide chamber at an appropriate temperature (26–37 °C, usually 26 °C) [28].

• 2. Fix the cells with 4 % PFA in 0.1 M PBS, pH 7.4 for 20 min (see Note 9).

• 3. Wash 3 × 10 min with 0.1 M PB.

• 4. Incubate the cells for 1 h in 5 % Block Ace with 0.01 % saponin in 0.1 M PB.

• 5. Apply the primary antibody (mouse anti-Yb, 1:250 dilution) for 24 h at 4 °C.

• 6. Wash 12 × 10 min with 0.1 M PB.

• 7. Apply the nanogold- and Alexa Fluor 488-conjugated anti-mouse secondary antibody (1:100 dilution) for 24 h at 4 °C.

• 8. Wash 12 × 10 min with 0.1 M PB.

• 9. Images can be captured on a fluorescence microscope during the PB washes of step 8 if desired (see Note 10).

• 10. Fix with 2.5 % glutaraldehyde.

• 11. Wash for 5 min with 0.1 M PB.

• 12. Wash 4 × 15 min with 50 mM HEPES Buffer, pH 5.8.

• 13. Wash 2 × 5 min with dH₂O.

• 14. Apply the silver enhancement solution from the HQ-Silver kit for 10 min in the dark room.

• 15. Wash 5 × 1 min with dH₂O in the dark room.

• 16. Wash for 5 min with 0.1 M PB.

• 17. Fix for 90 min with 1 % OsO₄ at 4 °C.

• 18. Dehydrate 2 × 5 min with 50 % ethanol at 4 °C.

• 19. Dehydrate 2 × 5 min with 70 % ethanol at 4 °C.

• 20. Dehydrate 2 × 5 min with 80 % ethanol at 4 °C.

• 21. Dehydrate 2 × 5 min with 90 % ethanol.

• 22. Dehydrate 2 × 5 min with 100 % ethanol.

• 23. Remove the line on the slide chamber glass, if necessary (see Note 6).

• 24. Apply the 100 % acetone for 5 min.

• 25. Apply the 100 % QY-1 2 × 5 min.

• 26. Apply the QY-1–Epon in a 1:1 ratio for 1 h.

• 27. Place the cells in 100 % Epon overnight.

• 28. Embed the glass slide in the silicon mold.

• 29. Incubate in 100 % Epon for 3 days (72 h) at 60 °C.

• 30. Store in a desiccator until dissection.

• 31. Remove the cells in Epon from the glass slides on top of a heat block at 120 °C.

• 32. Dissect the cells with an appropriate size (2 mm × 3 mm square).

• 33. Place the cells on a sectioning stage in a droplet of 100 % Epon.

• 34. Incubate overnight at 60 °C.

• 35. Store in a desiccator until sectioning.

• 36. Trim the block to an appropriate size (1 mm × 1.5 mm square).

• 37. Prepare the ultrathin sections (70 nm thick) with a diamond knife.

• 38. Collect the sections on a copper grid.

• 39. Dry the sections on the grid overnight.

• 40. Attach the copper grid to the grid stick for staining.

• 41. Incubate the sections in uranyl acetate for 10 min.

• 42. Wash the stick with the grids 3 × 1 min with dH₂O.

• 43. Incubate the sections in lead citrate for 10 min.

• 44. Wash the stick with the grids 3 × 1 min with dH₂O.

• 45. Remove the grid from the staining stick.

• 46. Dry the sections on the grid for 1 h.

• 47. Observe the cells by EM.

3.2 iEM Analysis for Tissue

• 1. Dissect out the target tissue.

• 2. Fix the tissue for about 10 h in 4 % PFA at 4 °C.

• 3. Incubate the tissue in 15 % sucrose in 0.1 M PB overnight at 4 °C.

• 4. Incubate the tissue in 30 % sucrose in 0.1 M PB overnight at 4 °C.

• 5. Immerse the tissue in cryocompound.

• 6. Freeze the tissue in an appropriate size cryomold with liquid N₂.

• 7. Prepare frozen 16–20 μm-thick sections of sample with a cryostat.

• 8. Dry the sections on glass slides for 1 h at 37 °C.

• 9. Wash the sections 3 × 10 min with 0.1 M PBS.

• 10. Incubate the sections in 5 % Block Ace with 0.01 % saponin in 0.1 M PB for 1 h.

• 11. Apply the primary antibody for 72 h at 4 °C.

• 12. Wash 10 × 10 min with 0.1 M PB.

• 13. Apply the nanogold-conjugated anti-mouse secondary antibody (1:100) for 24 h at 4 °C.

• 14. Continue the procedure with step 8 in Subheading 3.1.

3.3 EM-ISH Analysis for Cells

The representative images with this EM-ISH procedure are shown in Fig. 4.

• 1. Culture the cells (Drosophila OSCs) in a PLL-coated four-well slide chamber at an appropriate temperature (26 °C) (see Note 2).

• 2. Fix the cells with 4 % PFA in 0.1 M RNase-free PBS, pH 7.4, overnight at 4 °C.

• 3. Wash for 5 min with RNase-free 0.1 M PBS.

• 4. Wash for 5 min with RNase-free dH₂O.

• 5. Apply 0.2 N HCl for 20 min.

• 6. Wash for 5 min with RNase-free dH₂O.

• 7. Incubate in pre-warmed 100 mM Tris–HCl, pH 8.0, 10 mM EDTA at 37 °C.

• 8. Incubate the section in 1.5 μg/ml Proteinase K at 37 °C for 1 min (see Note 11).

• 9. Place slides in 0.2 % glycine/PBS for 10 min.

• 10. Wash for 5 min with RNase-free 0.1 M PBS.

• 11. Fix the cells for 20 min with 4 % PFA in 0.1 M RNase-free PBS, pH 7.4.

• 12. Wash 2 × 5 min with RNase-free 0.1 M PBS.

• 13. Wash for 5 min with RNase-free dH₂O.

• 14. Incubate for 15 min with Acetylation Buffer.

• 15. Wash for 5 min with RNase-free dH₂O.

• 16. Dip for 5 min in 100 % ethanol.

• 17. Air-dry.

• 18. Incubate the cells in prehybridization solution at 55 °C for 1 h.

• 19. Incubate the cells with FITC-conjugated specific RNA probe (1 μg/ml) for 16 h at 55 °C in hybridization buffer (see Note 12).

• 20. Wash 2 × 30 min with Wash Buffer 1 at 55 °C.

• 21. Wash for 5 min with NTET at 37 °C.

• 22. Treat for 30 min with RNase A (100 μg/ml) in NTET at 37 °C.

• 23. Wash for 5 min with NTET at 37 °C.

• 24. Wash for 30 min with Wash Buffer 2 at 55 °C.

• 25. Wash for 30 min with Wash Buffer 3 at 55 °C.

• 26. Wash for 5 min with TBS-T.

• 27. Apply Blocking Buffer for 1 h.

• 28. Apply primary antibody (rabbit anti-FITC, 1:500 dilution) for 24 h at 4 °C.

• 29. Wash 12 × 10 min in 0.005 % saponin containing 0.1 M PB.

• 30. Apply the nanogold- and Alexa Fluor 488-conjugated anti-rabbit secondary antibody (1:100 dilution) for 24 h at 4 °C.

• 31. Continue the procedure with step 8 in Subheading 3.1.

3.4 EM-ISH Analysis for Tissue

• 1. Prepare a fresh frozen tissue block.

• 2. Cut frozen sections (12–16 μm thick) with a cryostat and collect them on PLL- or MAS-coated glass slides.

• 3. Dry the sections for >2 h.

• 4. If the samples have been preserved in a −80 °C freezer, return them to room temperature without opening the slide case.

• 5. Edge each slide with silicon and dry it briefly.

• 6. Fix the tissues with 4 % PFA in RNase-free PBS for 30 min or at 4 °C overnight (use the latter for stronger fixation).

• 7. Wash the tissues for 5 min with RNase-free PBS.

• 8. Wash the tissues for 5 min with RNase-free dH₂O.

• 9. Apply 0.2 N HCl for 20 min, then briefly rinse with RNase-free dH₂O.

• 10. Incubate the section for 5 min in 3 μg/ml Proteinase K at 37 °C (see Note 11).

• 11. Transfer the slides slowly to a 0.2 % glycine/PBS solution and leave them for 10 min.

• 12. Wash for 5 min with RNase-free PBS, so as to not carry over any glycine to the next fixation step.

• 13. Post-fix for 20 min with 4 % PFA in RNase-free PBS.

• 14. Wash 2 × 5 min with RNase-free PBS.

• 15. Immerse briefly in RNase-free dH₂O, then incubate in Acetylation Solution.

• 16. Wash for 5 min with RNase-free dH₂O.

• 17. Dehydrate for 5 min with 100 % ethanol.

• 18. Dry the slides in a clean hood for 30 min.

• 19. Incubate the samples in prehybridization solution in a moisture chamber with swinging at 5 rpm at 55 °C for >30 min.

• 20. Hybridize with DIG- or FITC-labeled probes (final 2.5–10 μg/ml) in Hybridization Buffer in the moisture chamber with swinging at about 5 rpm at 55 °C for 16 h. To prevent the samples from drying up, cover the slides with a piece of Parafilm of the appropriate size.

• 21. Wash 2 × 30 min with Wash Buffer 1 at 55 °C.

• 22. Incubate for 15 min in NTET without RNase A at 37 °C.

• 23. Treat for 1 h with RNase A (10 μg/ml) in NTET at 37 °C.

• 24. Incubate the samples in NTET solution and leave them at 37 °C for 10 min. You can reuse the NTET solution from step 22.

• 25. Wash for 30 min with Wash Buffer 2 at 55 °C.

• 26. Wash for 30 min with Wash Buffer 3 at 55 °C.

• 27. Wash for 5 min in TBS-T.

• 28. Incubate for 30 min with Blocking Buffer.

• 29. Incubate the sections at 4 °C for 72 h with mouse anti-DIG (1:250 dilution) and rabbit anti-PSP1 (1:250 dilution) primary antibodies.

• 30. Wash 12 × 10 min in 0.005 % saponin containing 0.1 M PB at room temperature.

• 31. Incubate for 24 h at 4 °C with fluorescence- and nanogold-conjugated anti-mouse secondary antibodies (1:100) along with fluorescence-conjugated anti-rabbit secondary antibodies (1:800).

• 32. Continue the procedure with step 8 in Subheading 3.1.

4 Notes

• 1. For this iEM approach, any cultured cells are suitable: for instance, primary cultured cells or cell lines from human, marmoset, or rodent. This procedure is focused on adhesive cells cultured in chamber slides; however, the method is also suitable for free-floating cells or explant tissue cultures.

• 2. Regarding the chamber slides, a chamber with four wells is the most suitable for embedding, rather than a chamber with one, two, or eight wells.

• 3. 50–80 % ethanol solutions are stored at 4 °C, whereas 90–100 % ethanol is stored at room temperature.

• 4. Preparation of the plastic solution usually follows the manufacturer’s instructions.

• 5. The glass slide mold (microstar SNP-2) made of silicone should keep the glass slide or the chamber glass slide on the bottom of the mold until the resin has completely polymerized, to obtain an adequate thickness of plastic on the section.

• 6. A line-removal step using a single-edged razor blade is required for smooth removal of the resin-embedded cells from the slide chamber bottom, especially for chamber slides made of glass. This step is not necessary if using plastic-bottomed chamber slides. Lines from the PAP Pen or the Liquid Blocker for tissue sections should also be removed using a single-edged razor blade. Wear cut-resistant gloves for safety.

• 7. Both TEM and SEM are suitable for iEM and EM-ISH. For simplicity, this manuscript focuses on TEM. However, most of the materials and methods can be applied to various new serial-EM approaches, including the automated tape-collecting ultramicrotome-scanning electron microscope (ATUM-SEM), serial block face-scanning electron microscope (SBF-SEM), and the focused ion beam-scanning electron microscope (FIB-SEM).

• 8. Coating of the glass slides is critical for both iEM and EM-ISH to keep/remove the cells or tissue sections on the slides. Proteinase K or antigen-retrieval treatment will remove sections easily from a glass surface; however, the resin block preparation step requires a smooth peel-off (steps 31–33 in Subheading 3.1). A PLL- or poly-l-ornithine-coated slide chamber is appropriate for cultured cells, and MAS- or PLL-coated glass slides are suitable for tissue samples.

• 9. For general EM analysis (without immunostaining), fix the cells and tissues with 2 % glutaraldehyde, wash 3 × 10 min with buffer and restart from the secondary EM fixation with OsO₄ (step 17 in Subheading 3.1).

• 10. To capture the images with a fluorescence microscope, cover the cells/sections briefly with PB and carefully place a coverslip on top. An inverted microscope is not suitable because the coverslip will easily fall off.

• 11. The duration of the Proteinase K treatment step should be modified depending on the cell/tissue conditions (e.g., the cell type, fixation, and thickness of the sample). If the Proteinase K treatment is too strong to retain the normal membrane structure, reduce the concentration of Proteinase K solution, or substitute it with Triton™ X-100. As shown in Fig. 4, using Triton™ X-100 rather than Proteinase K better preserves the condition of the subcellular structures.

• 12. FITC-labeled RNA probes were prepared using RNA-labeling mixture and SP6 RNA polymerase according to the manufacturer’s instructions. To prepare a probe specific for the flam locus, OSC genomic DNA was used as a template for PCR.