Methods in Anopheles Research - MR4

Chapter 3 : Specific Anopheles Techniques3.1 Embryonic Techniques3.1.1 Microinjection Methods for Anopheles EmbryosPage 2 of 8• Double-sided adhesive tape. 3M type 415 has been tested for vertebrate toxicity and is a goodchoice (Method 1 only)• 22 x 22 glass or plastic coverglasses with a strip of pre-cut double-sided tape attached (Method 1only)• 25 mM NaCl (or 10-50 mM range for testing, Method 1 only)• Millipore (or other) mixed cellulose ester membranes (e.g. HAWP02500- US or HAWP03700,Method 2 only) 2Equipment• Either a compound or high-quality dissecting microscope can be used for injections. It ispreferable if it can be dedicated to this purpose.• Sutter P-2000 Micropipette puller or similar device 3• Needle positioner and holder• pH meter• Eppendorf Femtojet or similar device equipped with a foot pedal• Dissecting scope and illuminator for embryo alignmentSolutionsThis recommendation is for Drosophila melanogaster from Bill Engels lab, but it seems suitable formosquitoes. Prepare two 0.1 M solutions of monobasic and dibasic sodium phosphate. Mix the two andadjust pH with one or the other to pH 6.8-7.8. Prepare a solution of 0.5 M KCl in purified water.2X injection buffer is:0.2 mM Na phosphate10 mM KClFilter sterilize and store at room temperature or lower.Starting procedures common to both methods1. Prepare the capillaries by flushing them several times with purified water followed by ethanol toremove lint and glass chips. Blot the remaining ethanol and flame the capillaries briefly to remove allliquid. It is convenient to store them in a covered glass culture tube.2. Immediately before use, thaw the DNA and filter through a 0.2 micron Millipore Ultrafree-MC filterfilter to remove particulates. This latter measure (suggested by D. O’brochta) is simple and effective.Store on ice until use.2 Any non-fibrous membrane that is very thin, hydrophilic and does not contain detergent would probablywork for this e.g. Southern blotting membrane. If in doubt regarding the presence of wetting agents etc.rinse the membrane well before use. These types of membranes are perfect because when wet, theyadhere closely to a microscope slide so that the embryos don't slide beneath the membrane, and they areabout the same thickness as an embryo so visualization is easy.3 The program we use for the P-2000 is: HEAT: 650, FIL: 4, VEL: 40, DEL: 150, PUL: 157. However,conditions necessary to produce suitable needles may differ on your device and may require slightchange, even from day to day.