15 March 2007
FRANKS LAB - 1/11/07
PREPARING TISSUE FOR THE SEM
MATERIALS
Glutaraldehyde (25%) (cat# 18426 from Ted pella)
Osmium tetraoxide (cat#18451 from Ted pella)
NaH2po4
Na2HPO4
Scintillation vials.
Stock solutions
0.1M phosphate Buffer at pH 7.0 ( 200 ml)
39ml of 0.2M NaH2PO4
61ml of 0.2M Na2HPO4
100ml of dd water.
FIXATIVE SOLUTION
Working concentraion Amount (50ml) Stock solution
25mM Phosphate Buffer at pH 7 12.5 ml 0.1M Phosphate Buffer
3% Glutaraldehyde. 6.0 ml 25% Glutaraldehyde
31.5 ml dd water
PROCEDURE:
A. FIXATION -
Prepare fixative solution and fill the scintilation vials with fixative.
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cut tissue and place in tube with solution.
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Place tube on its side to keep the air away from the tissue.
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Incubate at 40C for 12-24 hours. ( Overnight is good).
B. OSMIUM TETROXIDE STEP -
Buy 0.5 gram capsules.
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Place 25 mLs of water in a bottle.
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Put capsule in bottle and break it opwn with a glass pipet. This makes about a 2% solution.
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Let solution sit at room temperature.
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The solution can be stored in the cold room or freezer for about 1 month. If frozen, thaw at room temperature. This solution should be straw colored. If it is purple it is no longer good.
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Dilute to a 1% solution in 25 mM phosphate buffer (25mM is the final concentration of PB!)
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Pour off fixative and add the 1% osmium tetroxide solution.
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Incubate in the cold room overnight to several days. The osmium turns black.
C. DEHYDRATION OF TISSUE
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Pour off osmium solution and rinse 3 times with 25mM PB. Be sure to put the first wash into the osmium waste.
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Put tissue through an alcohol series. 15 to 30 minutes each step.
a. 30% g. 100%
b. 50% h. 100%
c. 65% i. 100%
d. 75% J. 100%
e. 89% k. 2 more 100% soak next day
f. 95%
The tissue can be stored in 100% alcohol permanently.
D. CRITICAL POINT DRYING
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Put tissue into baskets.
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Fix with alcohol.
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Dry in the SEM facility.
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