del Carpio's Impregnation

for Reticulin on Previously Stained Sections

steps

materials

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Materials

Potassium permanganate, 0.25% aqu.
Oxalic acid, 1% aqu.
Silver nitrate, 2% aqu.
Silver nitrate, 10% aqu.
Sodium hydroxide, 40% aqu.
Ammonia, 20% aqu. (see note)
Formalin, 10% aqu.
Yellow gold chloride, 0.2% aqu.
Sodium thiosulphate, 5% aqu.
Neutral red, 1% aqu.
Ammoniacal silver

Solution Preparation

Place 1 mL of 10% silver nitrate in a flask.
Add 0.3 mL of 40% sodium hydroxide.
While swirling, slowly add drops of 20% ammonia until the precipitate just redissolves.
Make up to 100 mL with distilled water.

Tissue Sample

5 µ paraffin sections of neutral buffered formalin fixed tissue are suitable. Other fixatives are likely to be satisfactory. A section adhesive is recommended. The method was designed to demonstrate reticulin in sections which have been previously stained with dyes.

Protocol

Remove coverslip and bring slides to water with appropriate reagents.
Oxidise with 0.25% potassium permanganate for 20 minutes.
Rinse with tap water.
Place in Oxalic acid until bleached.
Wash with tap water.
Sensitise with 2% silver nitrate 24 hrs.
Rinse with distilled water.
Treat with ammoniacal silver for 30 minutes.
Rinse with distilled water, two changes.
Reduce in 10% formalin for 15 secs.
Rinse well with tap water.
Rinse well with distilled water.
Tone with 0.2% gold chloride solution.
Rinse with distilled water.
Fix in 5% sodium thiosulphate for 5 minutes.
Wash well with running tap water.
Counterstain with neutral red for 1 minute.
Rinse with tap water.
Dehydrate with ethanol, clear with xylene, and mount with a resinous medium.

Expected Results

Reticulin fibres – black
Nuclei – red
Background – grey

Notes

Strong ammonium hydroxide (s.g. 0.88) contains about 30% ammonia by weight. The instructions for making the ammoniacal silver solution specify a 20% ammonia solution, so strong ammonium hydroxide should be diluted about 2:1 with distilled water. Since the final step is to make up to 100 mL with distilled water, the precise concentration is not too important.
10% formalin is made by diluting strong formalin 1:10 with tap water (10 mL strong formalin, 90 mL tap water).
Toning is a variable step. Untoned sections give dark brown reticulin fibres on a paler brown background. Many microscopists prefer to tone for about 15 seconds to produce brown-black reticulin fibres on a pale grey-brown background. Others tone longer (a few minutes) to produce black reticulin fibres on a grey background. Longer toning produces purple tones. Tone according to the personal preference of the microscopist reviewing the slides.

Safety Note

Prior to handling any chemical, consult the Safety Data Sheet (SDS) for proper handling and safety precautions.

References

Gray, Peter. (1954)
The Microtomist’s Formulary and Guide.
Originally published by: The Blakiston Co.
Republished by: Robert E. Krieger Publishing Co.

del Carpio’s Impregnation for Reticulin on Previously Stained Sections