Autofluorescence
Cells and tissues can contain chemicals that are also excited by blue light. This is especially true of tissues such as the gut and yolk. Autofluorescence can usually be distinguished from GFP fluorescence because it is present across a much wider part of the spectrum. Thus, autofluorescence can usually be seen with a rhodamine TRITC filter set. With a long-pass emission filter, GFP will appear as a very characteristic shade of green, whereas autofluorescence will be yellow-green or yellow.
Method Freezing and Thawing EmbryosOneStep Procedure 31 Using the BioCool
1. Turn on COOL and PROGRAMMER, and turn magnetic stirrer all the way see Notes 1 and 2 . 2. Wait until the temperature comes up and green light is on. Hit PROG, and the number 1 appears. 3. Hit RUN the temperature will go to -6 C in about 20 min and hold there until straws are ready for up to 5 h . 4. Proceed to prepare the straws and the embryos. 5. When freezing is finished, hit ADV and the temperature will return to 20 C in about 30 min. 6. Hit RUN and turn off COOL, PROGRAMMER, and turn...
Fixation
1. To detect enzyme tracers or to cut tissue sections, embryos must be fixed in the appropriate solution. 2. If embryos are free-swimming tadpoles, anesthesize them before fixation by cooling on ice or adding several drops of MS222 to the culture medium. 3. Pick up embryos in a small volume of culture medium with a plastic transfer pipet and drop into a large volume of fixative in a vial with a tight-sealing cap. Use approx 40X vol of fixative per vol of embryo. 4. Place vials on a rotator for...
Instrumentation HardwareSoftware
1. Inverted epifluorescence microscope Zeiss axiovert 135, Carl Zeiss, Jena, Germany equipped with cover-slip-bottomed heating chamber Biophysica Technologies, Inc. , filters for epifluorescence Chroma Technology Corp., Brattleboro, VT and x10 Plan-Neofluar objective Zeiss, N.A. 0.3 . 2. PTI Deltascan dual monochromator illumination system with Ludl Electronic Products, Ltd. LEP , Hawthorne, NY, controller. 4. Slow-scan CCD camera CH250 Photometrics Ltd., Tucson, AZ 1.3 k x 1 k by 12 b . 5....
Notes Uqu
1. It has been found that sedatives may have different effects on embryonic mouse heart function during specific developmental time periods see ref. 8 . 2. Imaging at 7.5 MHz is sufficient to locate the embryo and the beating heart and to orient the sagittal plane of the embryo. This frequency is not adequate to directly image the internal anatomy of the heart. 3. The sample volume length is adjusted to 2-4 mm to completely insonate the contracting embryonic heart. The high-pass filter is at...
Microinjection
The microinjection technique is employed for the introduction of genes, radioactive precursors, cytoplasmic components, and cell lineage tracers into uncleaved eggs and early embryos Fig. 6A . A variety of specialized instruments and media are required in addition to the conventional operating tools Fig. 3 . 3.2.1. Injection System and Micropipets The injection system consists of a micromanipulator and an injector e.g., PLI-100, Medical Systems Co., Greenvale, NY . The volume of material...
Solutions for Fixation
1. For HRP-labeled specimens 4 paraformaldehyde, 1 glutaraldehyde, 0.5 dimethyl-sulfoxide DMSO in 0.1 M PB. Add 4 g paraformaldehyde to 40 mL dH2O. Stir constantly and heat to 60 C in a fumehood with the beaker covered with foil. Do not let the temperature rise above 65 C. Add 1 N NaOH dropwise until the solution clears. Cool solution on ice to room temperature. Add 2 mL of 50 EM-grade glutaraldehyde and 0.5 mL DMSO and mix thoroughly. Add 40 mL monobasic PB stock and 10 mL dibasic PB stock....
Materials Gth
a. Spearpoint microscalpel 6125 Roboz Surgical Instruments, Rockville, MD b. Two pairs of fine angled forceps Dumont 5-45 Fine Science Tools, Foster City, CA c. Miniature spatula Using a hammer and a sturdy block, flatten one end of a short length of 500- im diameter nickel-chromium wire Omega Engineering, Stamford, CT . Insert the round end of the wire into a pin holder 26016-12 Fine Science Tools and bend the flattened tip with forceps to a comfortable working angle approx 45 . d. Sharpened...
Making Chimeras by Morula Aggregation
The early morula is favored for making aggregation chimeras because blastomeres are both relatively adhesive and motile before the formation of junctional complexes between the outer, future trophectodermal, cells has progressed very far. The standard approach for producing such chimeras is to place developmentally synchronous, intact early morulae of different genotypes together in pairs, having first denuded them of the ZP. Successful aggregation leads to the formation of single integrated...
Phosphate Buffer Nutrient PB
1. To a 50-mL tube, add 30 mL of Dulbecco's PBS, 0.5 mL of pen strep solution, and 0.15 g of BSA. 2. Bring vol up to 50 mL with PBS and adjust pH to 7.2 if necessary. 3. Filter sterilize through 0.2-mm filter into a sterile tube. Store at 4 C. 4. Label and date for expiration 6 mo. Miscellaneous items ordered from IMV International Minneapolis, MN 1 2-cc straw cat. AAA101 , 1 4-cc straw cat. AAA201 , 13-mm goblets cat. PA003 , and 13-mm canes cat. XC055 . The liquid nitrogen LN2 dewar is...
Materials Fmx
1. Stereomicroscope, preferably with ground-glass stage, understage illumination, and up to at least 40X magnification, e.g., Wild M5A, Olympus SZH Olympus, Melville, NY . 2. 5 Watchmaker's forceps and small, sharp scissors. 4. 35-mm and 60-mm plastic petri dishes embryological watch glasses can also be used to collect the embryos. 5. Mouth-operated micropipet made from a drawn-out Pasteur pipet. 6. Flying saucer incubator Modular Incubator Chamber, Billups-Rothenberg, Del Mar, CA . 7. 5 CO2 5...
Embedding
Metal embedding molds are sprayed with mold release spray and preheated in the oven at 60 C. The molds are then filled with melted paraffin and kept on the hot plate of the embedding station. One at a time they are placed on the cold plate to allow the deepest portion of the mold to begin to harden they are then removed from the cold plate to a flat surface at room temperature. Heat lamps are placed immediately above the mold to keep the upper portion of the paraffin in its liquid state until...
Materials Miy
1. UBM Custom-built UBMs have been developed to perform the procedures described in this protocol 1,2 . Alternatively, it is possible to modify commercial high-frequency ultrasound scanners e.g., Ultrasound Biomicroscope, Paradigm Medical Industries, Salt Lake City, UT to perform in utero mouse embryo imaging Fig. 1A . In either case, the imaging system consists of a high-frequency 40-50 MHz focused ultrasound transducer that is scanned mechanically to produce two-dimensional 2-D images at...
Using a Confocal LaserScanning Microscope see Note 4
1. Yolk platelet cytoplasmic displacements Live embryos that had been incubated in Nile Red were placed in viewing dishes on the microscope stage between 15 and 20 min postfertilization in order to detect the earliest possible displacements of organelles. Because we did not use antifade reagents or other methods for reducing photobleaching, we used the fastest possible scan speed to establish the appropriate settings for data collection, e.g., gray-scale range, box size, and beginning and...
GFP cDNAs
The cDNAs for various GFP mutants are commercially available Clontech, Quantum Biotechnologies . These include both wavelength mutations and codon-optimized versions. Clontech and New England Biolabs Beverly, MA also supply anti-GFP antibodies. Antibodies are useful to determine whether any difficulties in visualizing GFP are at the level of protein expression or in the posttranslational cyclization. They can also be used to detect GFP after procedures that destroy its fluorescence, such as in...
SeongSeng Tan Leanne Godinho and Patrick P L Tam 1 Introduction
In female mammals, one of the two X chromosomes in each and every embryonic cell is randomly inactivated during embryogenesis, leaving only one active X chromosome per cell and thereby maintaining dosage parity with males 1 . This natural phenomenon, known as X inactivation or lyonization, provides an ideal nonsurgical method of producing mosaicism among otherwise identical cells of the embryo. We have created a line of mice H253 by pronuclear injection of a DNA fragment containing the lacZ...
Gastrulation in Reptiles and Birds
The eggs of reptiles and birds consist of a huge mass of yolk on which floats a small cytoplasmic cap consisting of thousands of blastomeres collectively forming the blastoderm. We discuss gastrulation in reptiles and birds together because gastrulation occurs similarly in these two classes of vertebrates. Gastrulation in reptiles and birds also involves epiboly, emboly, and convergent extension. Epiboly occurs as the extraembryonic, future yolk sac portion of the blastoderm grows downward and...
Info Ftk
Culture medium e.g., 1.5 ml embryo from GD 9 to GD11 Culture medium e.g., 1.5 ml embryo from GD 9 to GD11 Embryo within the yolk sac e.g., 3-5 embryos 60 ml bottle from GD 9 to GD 11 Fig. 15. Diagram of a culture bottle used for rat whole embryo culture. Fig. 16. Culture bottles that this author uses. Left Wheaton glass serum bottle Lawson Mardon Inc., Mays Landing, NJ . 50-mL size 223745 autoclave before use. Between three and five embryos can be cultured in a bottle. At least 4.5 mL of...
Adam S Doherty and Richard M Schultz 1 Introduction
The preimplantation mammalian embryo develops as a free living entity within the mother. This internal development inherently precludes facile experimental manipulation necessary to study cellular and molecular mechanisms of preimplantation development. In turn, this has led to intense efforts over the course of decades to develop culture media that support the preimplantation development in vitro and, in particular, mouse preimplantation development. By the mid-1960s and early 1970s, these...