Zenkers Fixative

1. Indications. Similar to Helly's fixative. Recommended for staining of cytoplasmic inclusions and for use with the Feulgen stain.

2. Composition. Stock solution: 50 g mercuric chloride (HgCl2), 25 g potassium dichromate (K2C2O7), 10 g sodium sulfate, anhydrous (Na2SO4), 1,000 mL distilled water.

Just before use, the stock solution is mixed with either acetic acid or formic acid: 95 mL stock solution, 5 mL glacial (99.7%) acetic acid, or 95 mL stock solution, 5 mL formic acid (88%, analytical grade).

3. Procedure. Fix slices not thicker than 6 mm in 20 volumes of fixative. The fixation time will be about 24 h. Thick specimens should be postfixed for 2 h in a 2.5% aqueous solution of potassium dichromate. Tissues must then be washed for 14-16 h. Transfer to 80% alcohol. Residues of mercuric chloride must be removed from the section with 0.5% aqueous iodine (5 min) followed by 5% aqueous sodium thiosulfate (5 min).

4. Storage. Use 70% ethyl alcohol for short-term storage. For long-term preservation, dehydration and paraffin embedding is recommended.

FIXATION BY MICROWAVE HEATING This technique has been used successfully both for light and electron microscopy (5). Heating of samples in saline to 58°C leads to good fixation for routine light microscopy but also for immunohis-tochemical reactions. For electron microscopy, samples should be placed in 2.5% glutaraldehyde and irradiated for 90 s to achieve a temperature of 58°C.

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