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TitleDevelopment of Sea Urchins, Ascidians, and Other Invertebrate Deuterostomes: Experimental Approaches
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Methods in Cell Biology

VOLUME 74

Development of Sea Urchins, Ascidians, and Other Invertebrate

Deuterostomes: Experimental Approaches

Page 454

j. When amyl acetate is used as a transition fluid, extensive washing (2–3 h) with

liquid CO2 is required to remove the amyl acetate before critical point drying. This

is costly in CO2. If ethanol is to be used as the intermediate fluid, the specimens

must be transferred rapidly to the apparatus and must be quickly immersed in

liquid CO2. Speed is necessary so as to prevent air-drying. As has been noted, the

use of the Teflon microporous capsules helps to retain some ethanol and prevent

air-drying during transfer. Only minimal washing with CO2 is required (circa

5 min) if the ethanol CO2 method is used.

k. Once the specimens have been dried, they can be transferred with a fine camel--

hair brush to an SEM stub coated with double-sided tape. The specimens can either

be coated whole or dissected with fine glass or steel needles prior to coating. In order

to achieve good grounding of the specimens, the edge of the tape should be over-

lapped with silver paint (Marivac, Halifax, NS). The thickness of the coating will

depend on the type of machine used and the magnification that is desired. The

coating should be as thin as possible, since thicker coatings obscure fine details.

VI. Quick Freezing and Freeze-Substitution

In most light and electron microscopic studies, chemical fixatives containing

formaldehyde, glutaraldehyde, and/or osmium tetroxide are used to prepare the

materials (Hayat, 1981). Ultrastructural studies often use several diVerent fixatives

as the morphology often varies somewhat, depending upon the technique

employed. These chemicals are thought to act by causing molecular cross-linking,

thus stabilizing molecules that may otherwise be extracted or relocated during

dehydration and infiltration of the tissues. While these techniques give excellent

morphological preservation, they tend to alter or destroy the immunological char-

acteristics of many compounds, denature proteins, and rearrange cellular and

extracellular components. In the last 25 years, more and more studies depend on

the use of immunological techniques (Hayat, 1981). In order to use immunological

techniques eVectively, preservation techniques must be developed that minimize or

eliminate the alterations or artifacts caused by chemical fixation (Fig. 4).

Rapidly freezing cells, tissues, or small embryos is one method that could

significantly reduce such artifacts. To achieve this, the tissues and/or embryos

are frozen so rapidly that all cellular processes are stopped instantaneously. In

order to achieve this, very small pieces of tissue were slammed on to copper blocks

cooled with liquid helium (Robards and Sleytr, 1985) by plunging them into

supercooled liquids such as liquid ethane or propane (Campbell et al., 1991) or

preserving them by a high-pressure freezing technique. This latter technique is

excellent but it involves the use of equipment that is expensive to buy and to use.

This then preserves the cells and tissues in a lifelike state without using aldehydes

and heavy metals. Nonaqueous solvents such as ethanol are then substituted for

the water ice and these, in turn, are used as a vehicle to infiltrate the tissue with a

fixative, if desired, and a resin.

428 Bruce J. Crawford and Robert D. Burke

Page 455

Experiments with starfish and sea urchin eggs/embryos/larvae (Campbell et al.,

1991; Crawford et al., 2000; Pang et al., 2002, 2003; Reimer and Crawford, 1995)

have shown that rapidly plunging the specimens into liquid propane gives good

preservation of the eggs/embryos/larvae, particularly those closest to the cryogen.

SEM Flow Chart

17. TEM and SEM Methods 429

Page 908

Volume 50 (1995)

Methods in Plant Cell Biology, Part B

Edited by David W. Galbraith, Don P. Bourque, and Hans J. Bohnert

Volume 51 (1996)

Methods in Avian Embryology

Edited by Marianne Bronner-Fraser

Volume 52 (1997)

Methods in Muscle Biology

Edited by Charles P. Emerson, Jr. and H. Lee Sweeney

Volume 53 (1997)

Nuclear Structure and Function

Edited by Miguel Berrios

Volume 54 (1997)

Cumulative Index

Volume 55 (1997)

Laser Tweezers in Cell Biology

Edited by Michael P. Sheez

Volume 56 (1998)

Video Microscopy

Edited by Greenfield Sluder and David E. Wolf

Volume 57 (1998)

Animal Cell Culture Methods

Edited by Jennie P. Mather and David Barnes

Volume 58 (1998)

Green Fluorescent Protein

Edited by Kevin F. Sullivan and Steve A. Kay

Volume 59 (1998)

The Zebrafish: Biology

Edited by H. William Detrich III, Monte Westerfield, and Leonard I. Zon

Volume 60 (1998)

The Zebrafish: Genetics and Genomics

Edited by H. William Detrich III, Monte Westerfield, and Leonard I. Zon

Volume 61 (1998)

Mitosis and Meiosis

Edited by Conly L. Rieder

882 Volumes in Series

Page 909

Volume 62 (1999)

Tetrahymena thermophila

Edited by David J. Asai and James D. Forney

Volume 63 (2000)

Cytometry, Third Edition, Part A

Edited by Zbigniew Darzynkiewicz, J. Paul Robinson, and Harry Crissman

Volume 64 (2000)

Cytometry, Third Edition, Part B

Edited by Zbigniew Darzynkiewicz, J. Paul Robinson, and Harry Crissman

Volume 65 (2001)

Mitochondria

Edited by Liza A. Pon and Eric A. Schon

Volume 66 (2001)

Apoptosis

Edited by Lawrence M. Schwartz and Jonathan D. Ashwell

Volume 67 (2001)

Centrosomes and Spindle Pole Bodies

Edited by Robert E. Palazzo and Trisha N. Davis

Volume 68 (2002)

Atomic Force Microscopy in Cell Biology

Edited by Bhanu P. Jena and J. K. Heinrich Hrber

Volume 69 (2002)

Methods in Cell–Matrix Adhesion

Edited by Josephine C. Adams

Volume 70 (2002)

Cell Biological Applications of Confocal Microscopy

Edited by Brian Matsumoto

Volume 71 (2003)

Neurons: Methods and Applications for Cell Biologist

Edited by Peter J. Hollenbeck and James R. Bamburg

Volume 72 (2003)

Digital Microscopy: A Second Edition of Video Microscopy

Edited by Greenfield Sluder and David E. Wolf

Volume 73 (2003)

Cumulative Index

Volumes in Series 883

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