TISSUE PROCESSING

Steps:

1. Fixation

5. Sectioning

2. Dehydration
6. Dewaxing

3. Clearing
7. Hydration

4. Infiltration/Embedding
8. Staining

1. FIXATION
The purpose is to preserve tissues permanently in as life-like a state as possible. Fixation should
be carried out as soon as possible to prevent autolysis. Fixation is best carried out close to neutral pH. The volume of fixative is important. There should be a 10:1 ratio of fixative to tissue.
Common Fixative:
10% Neutral Buffered Formalin:
Preparation of 1 litre:

Sodium phosphate monohydrate

Dibasic sodium phosphate anhydrate
37% Formaldehyde
Distilled water

- 4 gms
- 6 gms
- 100 ml
- 900 ml

2. DEHYDRATION
This is to remove the water from the tissues. This is usually done with a series of upgrading alcohol: 70% to 95 % to 100% ethanol. The duration of time of immersion in the alcohol depends on
the size of the tissues.

Large Tissues

Small Tissues

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50% ethanol
70%
80%
95%
100% I
100% II

1 hr
1 hr
1 hr
45 mins
45 mins
45 mins

45 mins
45 mins
45 mins
30 mins
30 mins
30 mins

3. CLEARING
This consists of removal of the dehydrant with a substance that will be miscible with the embedding medium (wax). The commonest clearing agent is Xylene. For best microscopy, the use of
cedarwood oil is recommended, though it is expensive.

Absolute: Cedarwood
Pure Cedarwood oil

- 6 hrs
- > 2 days

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half-day
> 2 days

4. INFILTRATION/EMBEDDING
Finally, the tissue is infiltrated with the embedding agent, almost always paraffin. A product called
paraplast contains added plasticizers that make the paraffin blocks easier to cut.