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Mounting embryos in permount
Chemical safety: You should wear gloves when handling any chemical. If the chemicals or solutions used in this lab come in contact with your eyes or skin, you should flush the affected area with copious amounts of water, and tell me about the spill. If a chemical comes in contact with your eyes, or if you have any ill effects from contacting a chemical, you should call a physician.
Mounting in permount: Mounting in permount is the best method for fixed, stained, gastrula stage embryos. This is because this method clears the yolk, making it much easier to visualize the staining. It is much messier and smellier and fussier than mounting in glycerol or methylcellulose.
1. Place embryo into microfuge tube. Remove most of the aqueous solution. Add about 1 ml of methanol and incubate with shaking for 10 min-overnight.
2. Remove the methanol, and add about 0.5 ml methyl salicylate. Incubate seconds to minutes, just until embryo sinks (seconds to a minute if possible depending on the age of the embryo). This is very smelly stuff. WEAR GLOVES. If you leave the embryo in this solution too long, the color will leach out.
3. Take a bridged cover slip of appropriate height. For gastrula stage embryos, use a three or four high cover slip. Place an "H" of permount on the slide (two lines on the border between the bottom slide and the cover slips making the bridge, and one across the middle). Place embryo in the middle of the line across the H (will be in the middle of the depression). Gently lay a 22X60 cover slip on top. Gently roll the cover slip to get the embryo in the right position. The permount will harden over the first ten minutes or so, so you want to do this fairly quickly. Notes of caution: use a separate pipet for each solution (aqueous, methanol, methyl salicyclate, ...). They do not mix well.
4. After your embryos have been mounted, keep them fairly dark. The yolk will turn pink with exposure to light. You want to take the pictures fairly quickly after you have mounted the embryos. They tend to turn colors and look ugly with the passage of time.