Gluck's Impregnation
for Reticulin on Zenker Fixed Tissue
Materials
- Oxalic acid, 5% aqu.
- Silver nitrate, 10% aqu.
- Strong ammonium hydroxide (s.g. 0.88).
- Sodium hydroxide, 40% aqu.
- Formalin, 5% aqu.
- Yellow gold chloride, 0.2% aqu.
- Neutral red, 1% aqu.
- Gram’s iodine.
Material Amount Iodine 1 g Potassium iodide 2 g Distilled water 300 mL Mix the iodine and potassium iodide in a 500 mL flask. Add 5 mL of the water. When the iodine has dissolved, make up to 300 mL with distilled water.
- Cajal’s Solution
Material Amount Strong formalin 15 mL Ammonium bromide 2 g Water 85 mL
Preparation of Gluck’s Ammoniacal Silver
- Place 10 mL of 10% silver nitrate in a flask.
- Add 0.5 mL of 40% sodium hydroxide.
- Allow to settle, then decant the supernatent.
- Wash the precipitate, allow to settle, then decant a few times.
- While swirling, slowly add drops of strong ammonium hydroxide until the precipitate just redissolves.
- Dilute to 100 mL with distilled water, then add 2 mL pyridine.
Tissue Sample
5 µ paraffin sections of Zenker fixed tissue are suitable. Other fixatives are likely to be satisfactory.
Protocol
- Bring sections to water via xylene and ethanol.
- Rinse well with tap water.
- Place in Gram’s iodine for 4-8 hours.
- Rinsewith 70% ethanol.
- Rinse well with tap water.
- Bleach in 5% sodium thiosulphate for 5 minutes.
- Wash well with tap water.
- Place in Cajal’s solution at 37°C for 24 hours.
- Wash with tap water.
- Place in Gluck’s ammoniacal silver for 5 minutes.
- Rinse with distilled water.
- Place in 5% formalin for 5 minutes.
- Wash with tap water.
- Tone with 0.2% gold chloride solution until grey.
- Rinse with distilled water.
- Place in 5% sodium thiosulphate for 5 minutes.
- Wash well with 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
- Ensure that both the ammonium hydroxide and sodium hydroxide are fresh and full strength. Keep both well stoppered when not in use. For the ammonium hydroxide, pour sufficient for use from the stock bottle into a beaker, then immediately restopper the stock bottle. Do not return excess ammonium hydroxide to the stock bottle.
- After making the ammoniacal silver solution but before adding the pyridine, smell the solution to ensure it has only a faint smell of ammonia. If the smell of ammonia is strong it indicates that too much ammonium hydroxide has been added. If so, it is preferable to make the solution again. Improperly made ammoniacal silver solutions can affect the quality of the impregnation.
- The formalin used to make Cajal’s solution should be neutralised, but do not use buffered formalin. Neutral formalin in this context may be made by keeping strong formalin over marble chips. However, be very careful as the gas given off may increase the pressure inside the container and cause an explosion. Either apply a cap loosely so gas can escape, or use a fermentation lock.
- 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.
