Staining Whole Mouse Embryos for β-Galactosidase (lacZ) Activity
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Staining Whole Mouse Embryos for β-Galactosidase (lacZ ) Activity
Andras Nagy , Marina Gertsenstein , Kristina Vintersten , and Richard BehringerThis protocol was adapted from "Techniques for Visualizing Gene Products, Cells, Tissues, and Organ Systems," Chapter 16, in Manipulating the Mouse Embryo , 3rd edition (eds. Nagy et al.). Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA, 2003.
INTRODUCTION
Whole mouse embryos are stained for β-galactosidase (lacZ ) activity using X-gal.
RELATED INFORMATION
A protocol for Staining Frozen Mouse Embryo Sections for β-Galactosidase (lacZ) Activity is also available.
MATERIALS
Reagents
Detergent rinse
Embryo staining solution
If staining is to be performed for more than 1 hour, add Tris-Cl (pH 7.3) to the staining solution to a final concentration of 20 mM.
Ethanol (optional; see Steps 4 and 5)
Fixative for staining intact embryos, freshly prepared
Mouse embryos (13 dpc or older) or pregnant mice (for embryos 15 dpc or younger)
Nuclear fast red (optional; see Step 5)
Paraformaldehyde (0.4%, w/v) in 1X PBS (optional; see Step 2)
Xylene (optional; see Step 5)
Wax (optional; see Step 6)
Equipment
Dissecting tools
Incubator, preset to 37°C
Microscope (optional; see Step 6)
Pasteur pipette (optional; see Step 2)
Tissue culture dish, 24-well (optional; see Step 2)
METHOD
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1. Dissect embryos free of their extraembryonic membranes.
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i. For embryos 13 days or older, slice them in half sagittally with a razor blade to facilitate penetration.
- ii. For embryos from 15 dpc to birth, it is advisable to perfuse the pregnant mouse.
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i. For embryos 13 days or older, slice them in half sagittally with a razor blade to facilitate penetration.
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2. Fix the embryos in fixative solution for best results. When examining many embryos at one time, place them individually in wells of a 24-well tissue culture dish and aspirate the fixative solutions with a Pasteur pipette. The fixation time depends on the size of the sample:
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i. Fix tissue culture cells for 5 minutes.
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ii. Fix early postimplantation embryos for 10-15 minutes.
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iii. Fix embryos up to ~13 dpc for 15-30 minutes.
If lacZ histochemistry is to be followed by antibody staining, a 0.4% paraformaldehyde fixation solution can be used instead.
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i. Fix tissue culture cells for 5 minutes.
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3. Rinse the fixed tissues in detergent rinse three times for 15-30 minutes at room temperature.
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4. Incubate the samples in embryo staining solution. The staining time depends on the size of the sample and the level of bacterial β-galactosidase activity:
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i. Incubate small samples in embryo staining solution at 37°C for 1-3 hours in the dark.
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ii. Incubate older embryos in embryo staining solution at 37°C for 4-5 hours or longer in the dark.
At this stage, embryos may be stored at 4°C in 70% ethanol. To image whole embryos, please see Imaging X-gal-Stained Mouse Embryos . To section embryos and observe lacZ expression at the cellular level, proceed to Steps 5-6.
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i. Incubate small samples in embryo staining solution at 37°C for 1-3 hours in the dark.
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5. After staining, dehydrate the embryos through a graded series of ethanol (70%, 90%, 95%, and 100%, prepared in xylene).
Do not leave the samples in alcohols and solvents longer than necessary because they may leach out the reaction product. Nuclear fast red can be used as a counterstain.
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6. Embed the embryos in wax, section them (see Sectioning Mouse Embryos , and view them under a microscope.
When sections are viewed by dark-field microscopy, the lacZ stain is pink and the contrast with surrounding tissues is good; therefore, no counterstaining is necessary.
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