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From Valentine et al, 1968. Biochemistry 7:2143-52

Rationale:

For the highest resolution with negative staining, there should be little or no support film, but some support is necessary to hold the protein. In this method the proteins are supported by a carbon film and "embedded" in a film of uranyl acetate. The film is cast on mica, which provides the cleanest possible surface for the carbon.

1. Freshly cleave a piece of mica, coat with a carbon film using the vacuum evaporator.

• Put ~30 mg/ml protein solution in a small vessel.
• Cut the mica to 3-4 mm2 pieces. Hold a piece with forceps and push into the solution of protein at a 30-45 degree angle. Do not let the film detach completely from the mica.
• Let the film sit on the protein solution for 20-40 seconds.
• Pull the mica back and allow the film to sit on the mica.

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Author: Microscopy Laboratory

General notes: The same procedures are used to fix and stain cells for SEM and for TEM. Cells can be fixed using conventional glutaraldehyde-osmium fixation described for transmission electron microscopy. To preserve ciliary orientation, use the "instant fixation" protocol described here. With this method, the cortex and ciliary beat form is well preserved but the cytoplasm is poorly preserved and membrane breakage and blebbing on the cell surface is evident.

To suspend cells, use a wide bore pipette and gentle flow to avoid damaging cells. Polypropylene disposable Pasteur pipettes work very well for all procedures.

Carry out all fixation procedures in a properly vented fume hood.

Supplies and most EM chemicals can be obtained from Electron Microscope Sciences, Ernest F. Fullum, Ladd Research Industries, Polysciences, Ted Pella, and Bio-Rad.

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Author: Microscopy Laboratory

The best way to fix a sample for electron microscopy is to follow a procedure developed and proven by others. If this is not possible, this method will produce good fixation for most tissues.

Solutions:

1. 100 mM PIPES, pH 7.0-7.4 or 50 mM Cacodylate, pH 7.4

• 2 mM MgSO4
• 0.25 M sucrose
• 1-2.5% glutaraldehyde
• 50 mM Cacodylate, pH 7.4 (note: cacodylate contains arsenic, so handle carefully)

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Author: Microscopy Laboratory

We recommend consultation with one of the lab directors before preparing specimens. The methods presented here provide an overview of preparation techniques for a variety of specimens.

• Conductive Specimens (such as metallic objects): Usually, these specimens do not have to be sputter coated. Simply mount the specimen on a SEM stub using conductive paint or putty.

• Non-conductive Dry Specimens (ex: ceramics, polymers): Mount on specimen stub with glue spots, double-stick tape, conductive paint or putty. These will need to be sputter coated.

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Author: Microscopy Laboratory

Method:

1. Pellet Tetrahymena cells in a clinical centrifuge. OPTIONAL: Suspend cells in HNMK (50 mM HEPES, pH 6.9, 36 mM NaCl, 0.1 mM Mg acetate, 1 mM KCl ) for 10-20 min at room temperture. This will remove precipitates present in the proteose peptone and provide cleaner surfaces if cells are processed for SEM.

• Pour off most of the medium and suspend cells in a slurry (<1 ml of remaining medium) at the bottom of the tube (be gentle).
• Fix by adding 5-10 ml of 2.5% glutaraldehyde in 100 mM HEPES, pH 7.2. Mix gently and let sit at room temperture for 1 hr.
• Gently pellet cells in the clinical centrifuge (don't pack cells). Suspend gently in 100 mM NaCacodylate, pH 7.2.
• Repeat step 4 three times.
• Prepare 0.5-1% OsO4 in NaCacodylate, pH 7.2 and add to the fixed cells - suspend gently and incubate on ice for 30-60 min.

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Abstract

1-cell embryo transfer is best performed after allowing injected embryos a little recovery time in culture. This allows better evaluation of the cells' survival - those that have been damaged during the injection process will undergo cytoplasmic condensation, causing the cellular material to become less glossy and darker in color as the cytoplasm shrinks away from the zona pellucida. This should be balanced against the increased survival rate with decreased in vitro exposure.

The Recipient

Careful selection of the recipient is most important as the pups are the end result of a lot of hard work. I personally use Swiss Webster mice, as they are quiet and make excellent mothers, although do become overweight quickly and exhibit bad planes of anesthesia when heavy. This is also a very inexpensive mouse to use. As an alternate, another strain I have used with considerable success is B6D2F1. These mice are hardy and display hybrid vigor.

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The aggregation method utilizes outbred blastocysts which are easier to obtain in numbers. The ES cells are intorduced into the developing embryo by the adherance of the cells to a dezonulated 8 cell stage embryo. The embryos are cultured to blastocysts and then transfered into a pseudopregnant hybrid (C57 X CBA) F1 recipient.

Method:

Preparation of the aggregation plate.

Aggregation and culture of the embryos with the ES cells is performed in microwells prepared in a plastic tissue culture plate using a darning needle.

1. In a 6cm petri dish add up to 10 drops of M16 media (ca. 3-4mm, larger allows too muh turbulence when the palte is moved).

• Cover the entire plate with parafin or Dow Corning Fluid 200 (viscosity 50cs).

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Method

This method works well for 17.5 day mouse embryos.

All stains should be made up fresh.

Fix embryos in 90% ethanol for at least 1 week, longer if possible. Prior to staining remove skin, and viscera, particularly the liver, kidneys and gut.

1. Add embryo to approx. 20mls alcian blue solution, leave for 3 days. The alcian blue may begin to precipitate out, just ignore this.

• Rehydrate as follows:

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Method:

Blastocyst transfer is usually performed 24 hours after aggregation when the morulae have become expanded blastocysts and on the same day as injection. A little time is given between injection and transfer to allow blastocysts to re-expand.

The Recipient

Careful selection of the recipient is most important as the pups are the end result of a lot of hard work. Two strains of mice are used:RB Swiss and (CBAC57BL6/J)f1. RB Swiss are quiet and make excellent mothers but they become overweight quickly and do exhibit bad planes of anaesthesia. CBAC57 mice are hardy and display hybrid vigour. They do not carry excess weight and go under anaesthetic well. This strain can be very nervous when housed separately which could be dangerous to their young. They are most suitable if a young RB Swiss is placed into the cage as a companion which can be removed as soon as the pups are seven days old. By this age destruction of the litter is very unlikely. If the CBA mother is to be housed alone she must no

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Before you start, have ready the following:

• Sterilize 2 pairs of curved forceps, 1 pair of iris scissors and 2 pairs of fine watchmaker forceps.
• 6ml syringe filled with DMEM with HEPES, with an 18 gauge needle attached. To the needle attach a 20cm length of clear vinyl tube and into the end of this tubing insert a sharp flusher.

At 2.5 days p.c. (post coitus) the morulae are present in the oviducts. For this reason, it is only necessary to remove the oviducts from the mouse.

Method:

1. Kill the 2.5 day pregnant mouse and lay on its back on benchcloth or absorbent paper.