dark because ammonia decolorizes them slightly. 6. Place in working solution of carmine for 20 to 30 minutes. Carry a control slide for checking. 7. Place for a few seconds in the differentiating solution.
Technique: Paraffin, sections to be cut at 6 microns.
Dissolve 1 g of hematoxylin in 22 cc of absolute alcohol in
an open dish on a hot plate. Cool, filter and add 8 cc of a
10% aqueous solution of ferric chloride and 8 cc of iodine
solution (2 g iodine, 4 g potassium iodide dissolved in
100 cc of distilled water). Always use freshly made
Acid fuchsin, aqueous solution 1% 5 cc
Saturated aqueous solution picric acid 100 cc
Sodium Thiosulfate (Hypo) Solution
1. As usual deparaffinize and take to water.
2. Verhoff’s elastic tissue stain for 15 minutes.
4. Differentiate in 2% ferric chloride__only a few minutes.
Check under microscope and if differentiated too far,
5. Place in 5% sodium thiosulfate for 1 minute.
6. Wash in tap water 5 minutes.
7. Counterstain in van Gieson’s stain for 1 minute.
8. Absolute alcohol—2 changes.
Elastic fibers—blue-black to black
Technique: Paraffin. Sections cut at 6 to 10 microns.
To 5 cc of 10.2% aqueous solution of silver nitrate, add
28% ammonia water, drop by drop, until the precipitate
which forms is almost dissolved. Add 5 cc of 3.1% sodium
hydroxide and barely dissolve the resulting precipitate with
a few drops of ammonia water. Make the solution up to 50 cc
with distilled water. Use at once. Glassware must be clean.
Neutral formaldehyde, 40% 0.5 cc
Uranium nitrate 1% aqueous solution
(Make fresh just before use) 1.5 cc.
[Break glass vial (15 grains) in graduated cylinder with
100 cc distilled water for 10% solution]
Sodium Thiosulfate (Hypo) Solution
Nuclear Fast Red (Kernechtrot) Stain
Dissolve 0.1 g nuclear fast red in 100 cc of a 5% solution of
aluminum sulfate with aid of heat. Cool, filter, add grain of
1. Xylene, absolute alcohol, 95% alcohol, distilled water.
2. Remove mercury precipitates if Zenker-fixed.
3. Wash well in distilled water.
4. Phosphomolybdic acid solution for 1 minute (oxidizer).
5. Rinse well in running water or cells will hold the
6. Dip in 1% aqueous uranium nitrate for 5 seconds or
7. Wash in distilled water for 10 to 20 minutes.
8. Place in ammoniacal silver solution for 1 minute.
9. Dip very quickly in 95% alcohol and go immediately
10. Reducing solution for 1 minute.
11. Rinse well in distilled water.
12. Tone in gold chloride solution for 1 minute or until
sections lose their yellow color and turn lavender.
Too much toning will make sections red. Check
individually under microscope.
14. Place in 5% sodium thiosulfate for 1 to 5 minutes.
16. Counterstain, if desired, with alum hematoxylin
and eosin, or nuclear fast red. Rinse well in distilled
18. Absolute alcohol—2 changes.
Other tissue elements__depending on counter-stain used.
Fontana-Masson Stain for Argentaffin Granules
Technique: Paraffin. Cut sections at 6 microns.
Silver Nitrate Solution (Fontana)
Dissolve 10 g of silver nitrate in 100 cc of distilled water.
To 65 cc of this solution, add ammonium hydroxide until
a clear solution with no precipitate is obtained. Add, drop
solution to cause the above solution to become slightly
cloudy. Let stand overnight before using. When ready
to use, dilute each 25 cc of silver solution with 75 cc of
Nuclear Fast Red (Kernechtrot) Solution
Dissolve 0.1 g of nuclear fast red in a 5% solution of
aluminum sulfate with the aid of heat. Cool, filter add a
grain of thymol as a preservative.
1. Deparaffinize and hydrate to water.
2. Immerse slides in silver nitrate solution for 1 hour at
4. Immerse in gold chloride solution for 10 minutes.
6. Place in sodium thiosulfate for 5 minutes.
8. Counterstain with eosin or nuclear fast red, if desired.
Argentaffin granules and melanin—black.
Technique: Cut frozen sections at 10 to 15 microns.
Heat until gelatin is dissolved. Add:
Harris’s Hematoxylin for counterstain
1. Carry sections through on an angled glass rod.
2. Dip sections in 70% alcohol for only a second.
3. Place in oil red O in a tightly closed container for 5
4. Wash quickly in 70% alcohol-avoid folds in sections.
6. Counterstain in Harris’s hematoxylin for a few
8. Blue in ammonia water. If sections are too dark
when removed from the hematoxylin, they may be
differentiated in 1% acetic water for a few seconds,
Best’s Carmine Stain for Glycogen
Fixation: Tissue must be fixed in absolute alcohol or
Carnoy’s fluid. Since glycogen is soluble in water go directly
from fixative to the clearing agent and then to paraffin.
Technique: Paraffin. Cut sections at 6 microns.
Boil gently and cautiously for several minutes. Cook in
open dish (evaporating dish). When cool, add 20 cc of
strong ammonia water. Keep in icebox.
2. Dip slides in very thin solution of celloidin—dry for a
3. Place in water to harden for a few seconds.
4. Stain in Harris’s hematoxylin solution for 15 minutes.
5. Differentiate in acid alcohol. Leave nuclei a little
dark because ammonia decolorizes them slightly.
6. Place in working solution of carmine for 20 to 30
minutes. Carry a control slide for checking.
7. Place for a few seconds in the differentiating solution.
8. Wash quickly in 80% alcohol.
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