FIXATION OF TISSUE FOR LIGHT MICROSCOPY

Bouin’s Fixative:

Picric Acid (saturated solution, aqueous) 75 ml

Formalin (concentrated, 37%) 25 ml

Glacial Acetic Acid 5 ml

Buffered Formalin, commercially available

1. Fix tissue in the fixative of your choice overnight at 4°C. Cut down pieces if they are very large

for better fixation.

2. Tissue can be stored in buffered formalin until you are ready to embed.

3. Wash tissue several times in 50% EtOH throughout the day to remove the picric acid. You may need to go longer if the tissue pieces are large.

4. Tissue can be stored in 70% EtOH overnight.

5. Dehydrate tissue through alcohol series as follows:

85% ~ 30 min to 90 min depending on size of tissue

95% ~ 2 changes; 30 – 90 min each

100% ~ 3 changes; 30 – 90 min each

1:1 EtOH:Xylene 2 changes; 30 - 90 min each

Xylene 2 changes; 30 – 90 min each

6. Transfer tissue to paraffin infiltrator and incubate under vacuum at 60°C overnight. Be sure

paraffin is fresh.

7. Place tissues back into vials and add fresh paraffin. Place in a 60°C oven for 2-4 hr.

8. Embed the tissue in paraffin blocks for sectioning.