The Harvard Medical School EM Facility is a fee-for-service core facility open to researchers in the HMS Quadrangle and the Longwood Medical area. The Facility provides services and supervision in Transmission Electron Microscopy.

Microscope sign up:

JEOL 1200EX - 80kV
Tecnai G² Spirit BioTWIN

Pricelist
Methods
Services
Equipment
Staff and how to contact us

Services

• Embedding specimens in resin
• Ultrathin sectioning of resin embedded specimens
• Ultrathin frozen sectioning (Tokayasu method)
• Immunolabeling of sections (resin and frozen)
• Freeze substitution followed by resin embedding (UV polymerization)
• Negative staining of small particles (e.g. purified viruses and proteins)
• Developing and printing of EM negatives
• Training in electron microscope operation

Pricelist

Prices take effect with December 2005 billing.

For more information about services or availability of equipment call 432-1698/1693 or E-mail maria_ericsson@hms.harvard.edu

Methods

• Fixation
• Embedding in resins
• Embedding in resins for immunocytochemistry
• Embedding in resins by freeze substitution
• Ultrathin frozen sectioning
• Immunogold labeling
• Negative staining

To be able to view a biological sample in the electron microscope it must first be stabilized or "fixed" in a way that the ultrastructure of the cells or tissue remain as close to the living material as possible. The choice of fixative depends on the purpose of your study. It should be noted that, even though standard protocols can be sufficient for many purposes, finding the right fixation conditions is often a matter of trial and error.

Glutaraldehyde
reacts with many nucleophiles in the cell (most commonly amines). Produces irreversible cross-linking networks throughout the cytoplasm in seconds to minutes. The reaction results in a drop in pH from a significant release of protons, making adequate buffering important. Note that a too high concentration of glutaraldehyde can inhibit the formation of rapid cross-links.

Formaldehyde
cross-links amino groups of proteins. The reaction is much slower than that of glutaraldehyde, normally using a 2-8% solution you need to fix for at least 2 hours at RT. Again, due to a drop in pH during the reaction of formaldehyde and amino groups adequate buffering is important.

Glutaraldehyde-formaldehyde mixtures
original paper by Karnovsky (1965) recommends a mixture of 4% glutaraldehyde and 6% formaldehyde. Other empirically determined mixtures are widely used for both structural and immunocytochemical studies. We routinely use a mixture of  2% formaldehyde and 2.5 % glutaraldehyde in 0.1 M Sodium Cacodylate buffer, pH 7.4.

Acrolein
very toxic, highly volatile reactive aldehyde that's mostly been used in mixtures with glutaraldehyde and/or formaldehyde. Cross-links proteins as rapidly as glutaraldehyde.

Osmium tetroxide
normally used as a secondary fixative after glutaraldehyde. Reacts with unsaturated acyl chains of membrane lipids and nucleophiles like amino and sulphhydryl groups. While osmiums main purpose usually is contrasting it also increases the retention of lipids in the tissue. Osmium is often destructive, affecting the structure of proteins by cleaving peptide bonds predominantly at tryptophane residues.

Tannic Acid
 a mixture of polyglycol anions that preserve tissue via non-covalent interactions. Can be used after primary fixation with an aldehyde to enhance contrast and preserve antigenicity.  Makes a good substitute for Osmium tetroxide when used in combination with 1% Uranyl Acetate.

Embedding in resins

The routine method for examining biological samples in TEM is to embed the material in plastic and cut ultrathin (~60-80nm) sections. The choice of resin depends on the type of sample and the purpose of your study. Listed below are some of the more commonly used ones.

Epoxy resins

Epon or Epon-Araldite mixtures are the most widely used resins for electron microscopy. Excellent for morphological studies, good cutting properties, stable under the electron beam. Not good for most immunocytochemistry purposes. Tissue has to be completely dehydrated in protein denaturing solvents (like propyleneoxide) before infiltration and polymerization has to take place at temperatures around 50-60° C. Epon is now sold under the name EMbed 812.

Spurrs is a Low Viscosity mixture which provides rapid infiltration of tissues. It's easy to prepare and mixes rapidly. Good cutting properties and stable under the electron beam. Compatible with ethanol so no change to propyleneoxide is needed prior to infiltration. Polymerization at 60°C is recommended.

Acrylic resins

LR White and LR Gold  are blends of hydrophilic and acrylic monomers that rapidly penetrate tissue because of their low viscosity. LR Gold is cured by UV-light in the cold while LR White can be cold cured (using an accelerator) or heat cured. The hydrophilic nature of these resins makes them usable for immunocytochemistry.

Lowicryl K4M, HM20, K11M and HM23 are highly cross-linked acrylate and methacrylate based embedding media designed for use over a wide range of embedding conditions. They are usable for embedding at low temperatures and immunocytochemistry.
 

Routine Protocol for Embedding in Resin

Our standard fixative is a mixture of  2% formaldehyde and 2.5 % glutaraldehyde in 0.1 M Sodium Cacodylate buffer, pH 7.4.

1.   Fixation of cells: Add fix 1:1 to the cell media. Leave for 1 hour at RT. If cells grow in suspension, spin them down in the fixative at 3000rpm for 3 minutes. Leave for 1 hour at RT.

      Fixation of tissue: Perfusion fixation is preferred. If this is not an option, immerse tissue pieces in 2% formaldehyde / 2.5% glutaraldehyde in 0.1M Sodium cacodylate buffer, pH 7.4 and fix for at least 2 hours at RT. Cut tissue into 1-2 mm cubes in the fixative.

2.    Wash in cacodylate buffer 3x
3.    1% Osmiumtetroxide/1.5% Potassiumferrocyanide (in H2O) leave for 1 hour at RT in the dark
4.    Wash in H2O (or in malelate buffer pH 5.15) 3-4x
5.    1% Uranyl Acetate in H2O  (or in maleate buffer) for 30 minutes
6.    Wash in H2O 3x
7.    Dehydration:       70% EtOH 15 min
                                  90% EtOH 15 min
                                 100% EtOH 2x15 min
8.    Propyleneoxide for 1 hour
9.    Infiltration: Epon mixed 1+1 with propyleneoxide RT 2-3 hours
10.  Move samples to embedding mold filled with freshly mixed Epon.
       Allow sample to sink, move to oven for polymerization.
11.  Leave to polymerize for 24-48 hours at 60°C.

Osmium Free Method for Resin embedding
(Tannic Acid / Uranyl Acetate method)

1.    Fix in PFA/ Picric acid.
       Do the following processing on ice until 100% Ethanol:
2.    1% Tannic Acid in Maleate Buffer (MB) for 40 min (make fresh TA every time)
       Rinse twice in MB
3.    In the dark: 1% Uranyl Acetate in MB for 40 min
       Rinse twice in MB
4.    Dehydrate 5 min each: 50%/70% Ethanol
5.    1% PPD(p-phenylenediamine) in 70% Ethanol for 15 min
        Rinse twice in 70% Ethanol
6.    Dehydrate 5 min each: 80%/95%/2x100% Ethanol
7.    Unicryl infiltration 2x1hour 1x Over night
8.    Fresh Unicryl : UV Polymerization at 4°C or in the oven at 40° 24-48 hours

AFS Freeze-Substitution Program for HM20

Program #0: (Substitution)
T1: -80°C for 36 hours in Methanol (precooled)
T2: -45°C for 1 hour  Preecool Resin Mixture
S1: +4°C/hour
Program#1: (Infiltration)
T1: -45°C for 1 hour  25% Resin/ 75%Methanol  (pre-cooled)
T2: -45°C for 1 hour  1:1 Mixture
T3: -45°C for 1 hour  3:1 Mixture
S1: O°C/hour
S2: 0°C/hour
Program#2: (Infiltration-cont.)
T1: -45°C for 1 hour  100% Resin
T2: -45°C Overnight  100% Resin
Program #3: (UV-Polymerization) T1: -45°C for 48 hours with UV-lamp on
T2:    0°C for 24 hours with UV-lamp on
T3:    RT for 2-3 days with UV-light on

Preparation of ultrathin frozen sections

Fixation of cells in culture: Attached cells: first remove cells from the dish with 5mM EDTA/PBS. Add cell suspension to a microfuge tube containing an equal volume 2x fixative (usually 4% PFA) in 0.1 M sodium phosphate buffer pH 7.4.

Cryoprotection: Small pieces (~2mm3) of tissue or cell pellet is infiltrated  in 2.3 M Sucrose in PBS containing 0.15M glycine. 3 drops for a total of 15-30 min at RT. (or overnight at 4 °C)

Freezing: Infiltrated tissue/cells are mounted on an aluminum pin, excess sucrose is removed with a filterpaper and the pin is plunged into liquid nitrogen.  Specimens can be stored for years under liquid nitrogen.

Sectioning: Ultrathin sections are cut at -120°C with a cryo-diamond knife, or at -90°C with a glass knife. Sections are picked up from the knife with a loop dipped a 2:1 mixture of  2.3M sucrose and 2% Methylcellulose and transferred to a formvar/carbon coated copper grid. Grids are left floating section side down on PBS or placed on 2% gelatin in a small petri dish and stored in the fridge until immunogold labeled.

Immunogold labeling procedure for Ultrathin Frozen Sections

if you stored the sections on 2% gelatin, warm up gelatin to 37degrees for 20 minutes, the gelatin will melt and you can  transfer the grids to drops of PBS. Wash on 4 drops of PBS before blocking in 1% BSA.

1. Blocking, 1% BSA in PBS                                                        15 min
2. 1° Antibody diluted in 1%BSA                                                  30 min
3. Wash 4 drops PBS                                                                   15 min
(4.) If 1° antibodies with weak binding capacity for protein A
 (mouse, rat, goat, sheep)  are used you need to use bridging
 antibody in 1% BSA (e.g. rabbit anti mouse)                                 30 min
5. Protein A-gold (PAG) in 1% BSA                                             20 min
6. Wash 4 drops PBS                                                                    15 min
In case of double labeling, fix in 1% Glutaraldehyde for 5 min
followed by quenching in 4 drops of 0.15 M Glycine/PBS and
repeat points 2-7 with another size PAG.
7. Wash on PBS                                                                              15 min
8. Wash 6 drops water                                                                     20 min

Contrasting procedure: Mix 9 parts 2% methyl cellulose with 1 part 3% aqueous uranyl acetate. Float grids on the mixture on ice for 10 min. Pick up with a 3.5mm loop and remove excess liquid with a filterable. Allow to dry before examining.

Negative Staining

The sample is diluted into water (if possible) and adsorbed onto a carbon or formvar/carbon coated grid. The carbon film becomes hydrophobic with time resulting in uneven spreading of the sample and the stain. The best result will be obtained if the grid surface is made hydrophilic prior to use.  This can be done with glow discharge in our vacuum evaporator. Once the specimen has been adsorbed onto the film surface, the excess sample is blotted off on a filterpaper (Whatman #1) and the grid is floated on a small drop (5 µl) of stain solution (most commonly 1-2% aqueous uranyl acetate or ammonium molybdate) for a few minutes and then blotted off. The sample is dried and examined in the TEM.
 

Equipment

• Reichert Ultratrim, for easy trimming of resin embedded specimens
• Nikon Eclipse E600 Microscope, for brightfiled and immunoflourescence
• Reichert AFS, Automatic Freeze-Substitution System
• Reichert KF-80 Cryofixation System, for immersion cryofixation and slam-freezing
• Pelco 3440 Laboratory Microwave Oven
• Reichert knifemaker

Staff

	Name	Telephone/ FAX	E-mail
	Maria Ericsson	617-432-1698 617-734-7557	maria_ericsson@hms.harvard.edu
	Louise Trakimas	617-432-1693	louise_trakimas@hms.harvard.edu
	Elizabeth Benecchi	617-432-1693	Elizabeth_Benecchi@hms.harvard.edu