Laboratory Protocols
Histological Staining Techniques
Gordon and Sweets' method for reticulin
Gordon & Sweets, 1936.
Gelatine coated slides recommended.
- Dewax sections, rinse in alcohol, rinse in water.
- Oxidise in acid potassium permanganate - 5 minutes.
- Wash briefly in water.
- Bleach in 1% oxalic acid - 2 minutes.
- Rinse in distilled water followed by a thorough wash in tap water.
- Sensitise in 2.5% iron alum - 10 minutes.
- Wash in several changes of distilled water.
- Treat with silver solution in a coplin jar - 5 seconds.
- Rinse briefly in distilled water.
- Reduce in 10% 'Analar' formalin in distilled water - 1 minute. (In fume hood.)
- Wash in tap water followed by distilled. (In fume hood.)
- Tone in 0.2% gold chloride - 2 minutes.
- Wash briefly in distilled water.
- Fix in 5% sodium thiosulphate - 2 minutes.
- Wash well in tap water.
- Rinse in distilled water.
- Counterstain with nuclear fast red - 5 minutes.
- Wash in distilled water.
- Dehydrate, clear and mount.
Results:
Reticulin - black.
Nuclei - red.
Collagen and cytoplasm - purple/pink.
Acid potassium permanganate
0.5% potassium permanganate in 3% sulphuric acidGold chloride = sodium tetrachloroaurate
Silver solution
To 5mls of 10% silver nitrate add strong ammonia drop by drop until the formed precipitate just redissolves. Shake the solution for a few seconds after adding each drop. Add 5mls 3% sodium hydroxide and redissolve the precipitate with a few more drops of ammonia. Again, shake the solution after adding each drop. The finished solution should have a slight opalescence. If the solution goes completely clear add a drop or two of 10% silver nitrate until a slight opalescence remains. Make up to 50mls with distilled water. Kept in a sealed coplin jar at 4oC the solution can be used for up to one month.
