Preparing tissue for the sem

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

1. 
   Prepare fixative solution and fill the scintilation vials with fixative.
   
2. 
   cut tissue and place in tube with solution.
   
3. 
   Place tube on its side to keep the air away from the tissue.
   
4. 
   Incubate at 4⁰C for 12-24 hours. ( Overnight is good).
   

B. OSMIUM TETROXIDE STEP

1. 
   Buy 0.5 gram capsules.
   
2. 
   Place 25 mLs of water in a bottle.
   
3. 
   Put capsule in bottle and break it opwn with a glass pipet. This makes about a 2% solution.
   
4. 
   Let solution sit at room temperature.
   
5. 
   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.
   
6. 
   Dilute to a 1% solution in 25 mM phosphate buffer (25mM is the final concentration of PB!)
   
7. 
   Pour off fixative and add the 1% osmium tetroxide solution.
   
8. 
   Incubate in the cold room overnight to several days. The osmium turns black.
   

C. DEHYDRATION OF TISSUE

1. 
   Pour off osmium solution and rinse 3 times with 25mM PB. Be sure to put the first wash into the osmium waste.
   
2. 
   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

1. 
   Put tissue into baskets.
   
2. 
   Fix with alcohol.
   
3. 
   Dry in the SEM facility.