用常规和体积电子显微镜对秀丽隐杆线虫胚胎进行超微结构分析的高压冷冻和冷冻替代的工作流程

Mohammad M. Rahman; Irene Y. Chang; Orna Cohen-Fix; Kedar Narayan

doi:10.21769/BioProtoc.3981

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A Workflow for High-pressure Freezing and Freeze Substitution of the Caenorhabditis elegans Embryo for Ultrastructural Analysis by Conventional and Volume Electron Microscopy

用常规和体积电子显微镜对秀丽隐杆线虫胚胎进行超微结构分析的高压冷冻和冷冻替代的工作流程

MR Mohammad M. Rahman ^* email

IC Irene Y. Chang ^*

OC Orna Cohen-Fix

KN Kedar Narayan email

(*contributed equally to this work) 发布: 2021年04月05日第11卷第7期 DOI: 10.21769/BioProtoc.3981 浏览次数: 5573

评审: Gunar FabigAnonymous reviewer(s)

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Abstract

The free-living nematode Caenorhabditis elegans is a popular model system for studying developmental biology. Here we describe a detailed protocol to high-pressure freeze the C. elegans embryo (either ex vivo after dissection, or within the intact worm) followed by quick freeze substitution. Processed samples are suitable for ultrastructural analysis by conventional electron microscopy (EM) or newer volume EM (vEM) approaches such as Focused Ion Beam Scanning Electron Microscopy (FIB-SEM). The ultrastructure of cellular features such as the nuclear envelope, chromosomes, endoplasmic reticulum and mitochondria are well preserved after these experimental procedures and yield accurate 3D models for visualization and analysis (Chang et al., 2020). This protocol was used in the 3D reconstruction of membranes and chromosomes after pronuclear meeting in the C. elegans zygote (Rahman et al., 2020).

Keywords: High-pressure freezing (高压冷冻)

Freeze substitution (冷冻置换)

C. elegans (秀丽隐杆线虫)

FIB-SEM (FIB-SEM)

Volume electron microscopy (体积成像电子显微镜)

vEM (vEM)

Background

C. elegans is a free-living nematode with many properties that make it amenable to scientific study: (1) the worms are ~1 mm long; (2) they are easy to grow, handle and maintain; (3) they proliferate rapidly and (4) they are amenable to genetic manipulations. The reader is encouraged to consult Corsi et al. (2015) for an excellent primer to C. elegans and its use as a model organism in biology. Embryonic cell division events in C. elegans are largely invariant both spatially and temporally, making the organism a robust eukaryotic model system (Oegema and Hyman, 2006). Transient changes in dynamic cellular components like the nuclear envelope and chromosomes during embryonic development can be well described at resolutions afforded by fluorescence microscopy (Cohen-Fix and Askjaer, 2017), but capturing the corresponding ultrastructural changes at higher resolutions, whether in two or three dimensions, is challenging (Altun et al., 2002). The chitinaceous shell of the embryos poses a diffusion barrier to chemicals, precluding conventional aldehyde-based fixation protocols; thus, rapid freezing of samples followed by freeze-substitution and imaging by EM is the preferred way to capture ultrastructural intermediates at nanometer resolutions. As these samples are too large for simple plunge freezing, they have to be high-pressure frozen, ideally in a manner conducive to screening for the correct developmental stage (Muller-Reichert et al., 2003; McDonald et al., 2010). Recently, we published a report describing architectural intermediates in nuclear envelope breakdown during embryonic development (Rahman et al., 2020). We visually followed C. elegans embryos trapped in capillaries until just before high-pressure freezing to ensure that the correct stages were frozen. In an accompanying methods paper, we also reported cryo-fluorescence microscopy of high-pressure frozen whole C. elegans worms followed by correlative FIB-SEM (Chang et al., 2020) to image such structures in the intact worm. In both these advances, central experimental steps included the appropriate high-pressure freezing, freeze-substitution and resin embedding of trapped worms and/or embryos. Here we provide a step-by-step protocol to correctly execute these procedures for downstream vEM analysis, either to replicate our findings, or to answer other questions of interest in C. elegans.

Materials and Reagents

C. elegans maintenance
1. Tissue culture dish, 35 × 10 mm (Corning, Falcon, catalog number: 353001 )
2. Worm pick (Genesee Scientific, catalog number: 59-30P16 )
3. Micropipette tips with barrier (any brand, 1 µl, 20 µl and 1,000 µl)
4. Serological pipets, 10 ml for OP50 seeding (Corning, Falcon, catalog number: 357551 )
5. C. elegans Bristol N2 [Caenorhabditis Genetics Center (CGC), https://cgc.umn.edu (Stiernagle, 2006)]
6. E. coli OP50 strain DA735 [Caenorhabditis Genetics Center (CGC), https://cgc.umn.edu]
7. Water, molecular biology grade (GE Healthcare, HyClone, catalog number: SH30538.02 )
8. Agar (RPI, catalog number: A20020-5000 )
9. Yeast extract (ThermoFisher Scientific, catalog number: BP9727-2 )
10. Bacto Peptone (BD, Bacto, catalog number: 211677 )
11. Sodium chloride (Avantor Performance Materials, J.T. Baker, catalog number: 3624-05)
12. Cholesterol (Sigma, catalog number: 1580-01 )
13. Ethanol 200 proof (Decon Labs, catalog number: 2716 )
14. Trizma-Cl (Roche, catalog number: 10812846001 )
15. Trizma-OH (Roche, catalog number: 10708976001 )
16. Luria-Bertani broth, bacterial culture medium (KD medical, catalog number: BLC-5020 )
17. M9 buffer (IPM Scientific USA, catalog number: 11006-517 )
18. Levamisole hydrochloride (Sigma, catalog number: 196142 )

High-pressure freezing and freeze substitution
1. Glass microscopy slides (ThermoFisher Scientific, catalog number: 10144633B )
2. Tubes, 1.5 ml for BSA solution aliquots (ThermoFisher Scientific, catalog number: 05-408-129 )
3. Micropipette tips with barrier (any brand, 1 µl, 20 µl and 1,000 µl)
4. Micropipette tips without barrier (Eppendorf, 1 µl, catalog number: 30072.014 )
5. Cellulose capillary tube (Leica, catalog number: 16706869 )
6. Needles, 21-gauge (G) x 1½ inch (Covidien Monoject, catalog number: 305167 )
7. Syringe, disposable (VWR, EMS, catalog number: 72508 for 2.5 ml or 72509 for 5 ml)
8. Alcohol swabs (BD medical, catalog number: 326895 )
9. Type A gold-coated copper planchette (Leica, catalog number: 16770152 )
10. Type B gold-coated copper planchette (Leica, catalog number: 16770153 )
11. Cryomarker, Black (ThermoFisher Scientific, Nalgene, catalog number: 22-026-700 )
12. Sample holder Cartridge system, D3 mm half cylinder (Leica, catalog number: 771849 )
13. Sample holder Cartridge system, D3 mm middle plate (Leica, catalog number: 771813 )
14. Nalgene cryovials (ThermoFisher Scientific, catalog number: 5000-1012 )
15. Polypropylene tubes, 50 ml (BD Falcon, catalog number: 352098 )
16. String or twine (VWR, Twine, catalog number: 30-33113 )
17. Disposable cellulose acetate filter, 0.22 µm mesh size, diameter 25 mm (Millipore, Cameo syringe filter, catalog number: 1213657 )
18. Erlenmeyer glass flasks, 250 ml (VWR, Pyrex, catalog number: 4444-250 )
19. Pasteur pipettes, Borosilicate glass, 5 ml (ThermoFisher Scientific, catalog number: 13-678-20A )
20. Pasteur pipettes, plastic, 2 ml and 7 ml (Globe Scientific, catalog numbers: 137040 and 134090 )
21. Serological pipette, Borosilicate glass, 1 ml (VWR, catalog number: 93000-682 )
22. Plastic cups (for weighing resin) (VWR, Therapak, catalog number: 74850 )
23. Beem capsules (Ted Pella, catalog number: 69910-01 )
24. Beem capsule holder (Ted Pella, catalog number: 132-B )
25. Double Edge Carbon steel blade (Ted Pella, Feather, catalog number: 121-9 )
26. Kimwipes 05517 (ThermoFisher Scientific, Kimberly-Clark Kimtech Science Precision wipes, catalog number: 06-677-72 )
27. Styrofoam container or tray (e.g., a shipping container lid, typically 15 × 20 cm and 2 cm deep)
28. Disposable polypropylene spatula for osmium handling (VWR, catalog number: 80081-194 )
29. Bovine Serum Albumin (BSA), heat shock fraction (Sigma, catalog number: A3294 )
30. Nail polish (any color)
31. Osmium tetroxide (OsO₄) granules (EMS, catalog number: 19134 )
32. Acetone (ThermoFisher Scientific, catalog number: 9011 )
33. Uranyl acetate (EMS, catalog number: 22400 )
34. Methanol (Mallinckrodt, catalog number: 3016 )
35. Poly/Bed 812 embedding kit with DMP-30 (Polysciences, catalog numbers: 0 8792 and 0 8791 )
36. Dry ice (in-house supply)
37. Double deionized water (in-house supply)
38. Dry liquid nitrogen (LN₂) (in-house supply)
39. Dodecenyl succinic anhydride (DDSA) (Polysciences, catalog number: 0 8792 , kit same as 35)
40. Nadic Methyl anhydride (NMA) (Polysciences, catalog number: 0 8792 , kit same as 35)
41. Modified Youngren’s, Only Bacto-peptone (MYOB) plates for worm maintenance (see Recipes)
42. Cholesterol stock solution (see Recipes)
43. E. coli OP50 stock (see Recipes)
44. Cellulose capillary attachment (see Recipes)
45. 20% BSA solution (see Recipes)
46. 25 mM Levamisole solution (see Recipes)
47. Quick Freeze-substitution (QFS) cocktail (see Recipes)
48. Poly/Bed 812 resin mix (see Recipes)

Equipment

Micropipettes (any brand, for 1 µl, 20 µl and 1,000 µl volumes)
Sharp-point tweezers (EMS, Dumont, catalog numbers: 78320-51T , 72919-0A , and 78340-51S ) (Figure 1a-1c)
Tweezers insulated with PVC (EMS, Dumont, catalog number: 3C-linox-E ) (Figure 1d)
Forceps insulated with PVC, long (Leica, VOMM, catalog number: 22SAESD ) (Figure 1e)
Crimping tool, scalpel #20 (Bard-Parker, catalog number: 371620 ) (Figure 1f)

Figure 1. Tools for sample handling. a-c. Sharp-point tweezers for sample handling (Section A Steps 4-5). d-e. Insulated tweezers for frozen sample recovery and transfer (Section B Steps 4-9, and Section C Steps 6-7). f. Crimping tool: #20 scalpel manually blunted by rubbing the blade gently on a metal surface (McDonald et al., 2010).
Table-top centrifuge (Eppendorf, model: 5424 or similar)
Bottom illuminated stereomicroscope with frosted glass stage (Leica, model: SM2745 or similar)
Leica high-pressure freezer, ultra-low temperature equipped with stereomicroscope and funnel to fill LN₂(Leica, model: ICE )
Frozen sample recovery cryobox (stainless steel tray, deep) with frozen sample release station and 3 mm punch, rod, and plug (Leica, Austria; EM ICE high-pressure freezer package)
Mini benchtop orbital shaker (VWR, catalog number: 97109-890 )
Large surface slide warmer (Premiere, catalog number: XH-2002 )
Infrared thermometer (General Tools & Instruments, catalog number: IRT207 )
Analytical balance (Sartorious Corp, model: BCE64-15 )
Stirring plate (Corning, catalog number: PC420D or similar)
Standard desiccator connected to a mechanical pump (Ted Pella, model: VRD4 )
Metal blocks with 12 mm holes (ThermoFisher Scientific, catalog number: 88880152 )
Dry ice buckets, multiple (VWR, Scienceware Magic Touch 2 with lid, catalog number: M16807-2001 )
Bunsen burner
Chemical safety hood
Laboratory timer
Chemical scale (Mettler, model: AE240 )
Temperature-controlled oven (Quincy Lab, model: 20GC )
Standard autoclave
Hairdryer (any brand with hot air fan)
Sample storage, large LN₂ dewar (Taylor-Wharton, catalog number: HC34 )
Portable LN₂ dewar on a rolling base, 25 L (Worthington, catalog number: LD25 )
Portable LN₂ dewar, 4 L (Worthington, catalog number: LD4 )
PPE (lab coat, face shield and cold-resistant gloves) for handling LN₂ (any brand)
Facemask, surgical (Halyard health, catalog number: 28806 ; or any brand)

Procedure

English

中文翻译

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如何引用

Readers should cite both the Bio-protocol article and the original research article where this protocol was used:

Rahman, M. M., Chang, I. Y., Cohen-Fix, O. and Narayan, K. (2021). A Workflow for High-pressure Freezing and Freeze Substitution of the Caenorhabditis elegans Embryo for Ultrastructural Analysis by Conventional and Volume Electron Microscopy. Bio-protocol 11(7): e3981. DOI: 10.21769/BioProtoc.3981.
Rahman, M., Chang, I. Y., Harned, A., Maheshwari, R., Amoateng, K., Narayan, K. and Cohen-Fix, O. (2020). C. elegans pronuclei fuse after fertilization through a novel membrane structure. J Cell Biol 219(2). doi:10.1083/jcb.201909137.