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MESAB
Tricaine: 3-amino benzoic acid ethyl ester from Sigma (Cat # A-5040).
Mix in fish safe container with a stir bar:
400 mg tricaine powder
800 mg Na2HPO4 (anhydrous)
100 ml glass distilled water
Adjust to ~pH 7 with a drop at a time of 1N NaOH or 1N HCl if needed but it's
usually right if you weigh the sodium phosphate carefully and measure the water with a
graduated cylinder.
For storage: Aliquot into 6 x 25 ml fish safe plastic bottles and store at 4C. Label
with date made and use within a couple of weeks.
Dilution for babies: 10 drops in fish water (water in which fish are kept) in a 35 mm
diameter disposable Petri dish. (~40-50 mls of liquid). Embryonic zebrafish can
withstand being in MESAB for hours or until the anesthetic wears off. This will slow
their growth rate, but does not seem to cause abnormal development of structures. Dilution for adults: 4.2 ml in 100 ml fish water in a beaker. Adults should be left in
beaker just until their breathing slows and they lay on their sides, then rinsed off in clean
fish water very briefly before examining or in vitro fertilization procedures. They should
be returned to clean water as soon as possible. Adult fish are very sensitive to anesthetic
and will die if left in it too long.
8% PFA
8 g Pelleted PFA (Ted Pella, Inc.; cat# 18501)
90 ml dH2O
25 drops 1N NaOH
Heat at very low heat and stir until sol'n clears.
Add 25 drops 1N HCl. pH should be 7.0-7.2.
Filter and store at 4C not more than 1 week.
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Alcian blue will not readily dissolve in 90%ETOH. To make 100 mls add 0.2g alcian blue 8GX powder Alcian Blue 8GX (Anatech, Ltd. Cat.# 862) to 11.2ml 50%ETOH. Warm and occasionally swirl. When all is dissolved add 95%ETOH to 100 ml. Check clarity of solution under a microcope to make sure there are no precipitates.
0.04% Alcian/10mM MgCl2 Stain pH 7.5
To make 50 mls:
10ml 0.2% alcian blue Alcian Blue in 90%ETOH
5ml 1M Tris pH 7.5 0.5ml 1M MgCl2
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5ml 1M Tris pH 7.5
31.8ml water
0.5% alizarin red Alizarin Red S Stock: To make 50 mls add 0.25g alizarin red S powder Alizarin Red S (JT Baker cat# A475-03) to 50 ml water.
0.01% Alizarin Stain, pH 7.5
To make 50 mls:
1ml 0.5% alizarin red SAlizarin stock
12.5ml 100% glycerol
5ml 1M Tris pH 7.5
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