Histological dyes 2
1. Aniline Blue-Orange G (according to Heidenhain a.o. for AZAN)[2]
- Heat 2.5gr Aniline Blue Orange G (1A-352[3]) and dissolve in 100ml AD;
- in the hot solution add 8ml glacial acetic acid 98%;
- after cooling, filter the solution.
2. Aniline Blue Orange G (according to Heidenhain a.o. for AZAN)[1].
- Dissolve 0.5gr Aniline Blue (soluble in water, 1B-501[3]) and 2gr Orange G (1B-221[3]) in 100ml AD;
- Add 8ml glacial acetic acid;
- boil solution and after cooling filter;
- dilute this stock colouring solution 1:1 to 1:3 with AD.
3. Aniline Blue Orange G (according to Mallory)[2].
- Dissolve 4.5gr Aniline Blue-Orange G (1A-550[3]) in 100ml AD by boiling;
- after cooling, filter the solution.
4. Aniline Blue-Orange G (according to Mallory)[1].
- Dissolve 0.5gr Aniline Blue (soluble in water, 1B-501[3]) and 2gr Orange G (1B-221[3]) in 100ml AD;
- Add 1gr Phosphorus Tungsten Acid (3D-092[3]);
- filter.
5. Azur-Eosine (Giemsa[2])
- Dissolve 0.76gr Azur-Eosine (1A-174[3]) in 50ml methyl alcohol (3E-158[3]) and 50ml glycerine (3D-086[3]) at 60°C;
- Methyl alcohol and glycerine must be absolutely acid and anhydrous;
- After cooling, filter the solution in an absolutely dry bottle.
6. Barlow's trichrome[2]
- Dissolve 1.55gr Barlow's trichrome (1A-522[3]) in 60ml AD by boiling;
- Add 0.25ml concentrated nitric acid (3L-053[3]);
- after cooling, add 60ml glycerin (3D-086[3]) and mix well.
7. Bismarck brown[1]
- Dissolve 0.5gr Bismarck brown (1B-261[3]) in 100ml of 30% methyl alcohol (3E-158[3]);
- Filter (do not boil or heat, otherwise the dye will disintegrate).
8. Borax carmine[2]
- Dissolve 4gr Borax carmine (1A-434[3]) in 100ml AD by boiling;
- Add 100ml Ethanol (3L-132[3]);
- after cooling, filter the solution.
9. Spectacle Cresyl Blue[2]
A, tribal solution;
- Mix 0.85gr sodium chloride with 100ml AD and 7gr brilliantcresyl blue (1B-519[3]);
- mix, do not heat.
B, usage solution;
- Mix 10ml stock solution with 40ml NACL solution of 0.85%.
10. Karbolfuchsine[1] (Spermatozoon)
- Dissolve 5gr Phenol (3L-001[3]) in 84ml AD;
- Add 1gr New fuchsine (1B-467[3]);
- after mixing, add 10ml ethanol.
11. Karbolfuchsine[2] (Ziehl-Neelsen)
- Dissolve 1.6gr Karbolfuchsine (1A-586[3]) in 90ml AD and 10ml ethanol while heating (do not boil);
- filter after cooling.
12. Karbolgentian violet[2](Gram staining)
- Dissolve 0.6gr Karbolgentian violet in 95ml AD and 25ml ethanol while heating (do not boil);
- filter after cooling.
13. Karbol Chromotrop 2R[1] (Cytology, granules of eosinophilic granulocytes)
- Melt 1gr Phenol (3L-001[3]) in a flask in a hot water bath;
- add 0.5gr Chromotrop 2R (1B-259[3]) and mix with the melted phenol;
- mix the solution with 100ml AD;
- the dye has a shelf life of 3 months.
14. Karmaluin[2] (Core colouring after silver plating techniques)
- Dissolve 5.6gr Karmaluin (1A-334[3]) in 100ml AD by boiling;
- filter after cooling.
15. Carmine according to Best[1] (Glycogen)
- 2gr Carmine (5A-380[3]) and 5gr potassium chloride (3G-034[3]) when heated dissolve in 60ml AD;
- Add 1gr of potassium carbonate (3G-033[3]) and boil gently (lather strongly);
- let boil for a few minutes. The color changes to dark red;
- after cooling add 20ml ammonia (3L-136[3]);
- store in tightly closed bottle in a cool place. The dye can be kept for about 2 months.
16. Carmine according to Best[2] (Glycogen)
- Dissolve 11.5gr Carmine according to Best (1A-358[3]) by boiling in 100ml AD;
- after cooling add 3ml ammonia (3L-136[3]).
17. Carmine glacial acetic acid according to Belling[1] (Chromosomes, smear preparation. Not suitable for archiving).
- Bring 100ml of 45 % ice vinegar to the boil;
- Add 0,5gr Carmine (5A-380[3]) and dissolve;
- Continue boiling (1-2 minutes) until the colour changes and darkens;
- after cooling, filter and store in a dark bottle.
18. Carmine glacial acetic acid according to Schneider[2] (Chromosomes, smear preparation. Not suitable for archiving).
- Mix 45ml glacial acetic acid (3D-087[3]) with 55ml AD;
- Add 0,5gr Carmine (5A-176[3]) and cook for 30min;
- filter after cooling.
19. Crystal Violet[2]
- Dissolve 2gr Crystal Violet (1B-345[3]) in 10ml ethanol and 90ml AD by heating (do not boil);
- filter after cooling.
20. Triacid according to Ehrlich[1]
- Grind 3,4gr methyl green (1A-292[3]), 4,2gr fuchsine acid (1B-525[3]) and 3gr orange G (1B-221[3]) in a mortar;
- Heat 100ml AD to 35°C and the mixture will ripen for 2-3 days under regular shaking;
- filter after cooling.
21. Triacid according to Ehrlich[2]
- 35ml Ethanol, 15ml glycerine (3D-086[3]) and 50ml AD heating;
- Dissolve 2gr Triacid powder (1A-572[3]);
- filter after cooling.
22. Orcein ice cream vinegar according to La Cour[1] (Chromosomes, smear preparation or paraffin coupes).
A, tribal solution;
- Heat 100ml glacial acetic acid to boiling point;
- Dissolve 2.2 g Orcein (1F-337[3]) and allow to cool.
B, use solution;
- Mix 4.5 ml stock standard solution with 5.5 ml AD and filter.
23. Iron haematoxylin according to Refuses[1].
A;
- Dissolve 1gr Haematoxylin (5B-535[3]) in 100ml slightly heated ethanol 96%;
- Allow to mature for 1 week. The solution has a practically unlimited shelf life.
B;
- Dissolve 1.5gr Iron (III) chloride (3L-003[3]) in 100ml AD and add 1ml concentrated hydrochloric acid (3L-012[3]).
Immediately before use mix solutions A and B 1:1
24. Ironhaematoxylin according to Refuses[2]
A;
- Dissolve 1gr Haematoxylin (5B-535[3]) in 100ml slightly heated ethanol 96%;
- Allow to mature for 1 week. The solution has a practically unlimited shelf life.
B;
- Dissolve 4ml Iron (III) chloride (3L-007[3]) in 95ml AD and add 1ml concentrated hydrochloric acid (3L-012[3]).
Mix solutions A and B 1:1 immediately before use.
25. Iron trixihaematin according to Hansens[2].
A;
- 10gr iron (III) ammonium sulphate (3G-095[3]) dissolve in 150ml heated AD.
B;
- Dissolve 1.6gr Haematoxylin (5B-535[3]) in 75ml heated AD.
As soon as the liquids have cooled to room temperature, mix solution A, stirring constantly, with solution B (not the other way around). The solution turns brown first, then blue and then dark violet.
Then heat the mixture until it has turned brown again. If the solution turns olive green, too much oxidation has taken place. Reduction is achieved by dripping a 10% oxalic acid (3G-068³) solution into the warm solution until the colour has turned brown again. After cooling the solution must react with acid. If necessary, acidify with oxalic acid.
26. Eosin aqueous[2]
- Dissolve 1gr Eosine (1B-425[3]) in 100ml heated AD;
- filter after cooling.
27. Eosin alcoholic[2]
- Dissolve 1gr Eosine BA (1B-421[3]) in 70ml methyl alcohol (3E-158[3]) and 30ml AD;
28. Eosine methylene blue according to May-Grünwald[2] (Blood staining)
- Dissolve 0.25gr Eosin-methylene blue (1A-172[3]) in 100ml heated methyl alcohol (3E-158[3]) at 60°C;
- after cooling, filter through a dry filter in a dry bottle;
- the solution has a good shelf life.
To guarantee a standard colouring, it is better to buy an industrially prepared solution.
For Chroma this is: 2E-008
29. Eosine methylene blue according to Wright[2] (Blood staining)
- Dissolve 0.2gr Eosin-methylene blue (1A-324[3]) in 100ml heated methyl alcohol (3E-158[3]) at 60°C;
- after cooling, filter through a dry filter in a dry bottle;
- the solution has a good shelf life but should not be near acidic solutions.
To ensure a standard colouring, it is better to purchase an industrially prepared solution.
For Chroma this is: 2E-036
30. Gentian violet according to Flemming[2] (Gram)
- Dissolve 1gr Gentian Violet (1B-411[3]) in 20ml methyl alcohol (3E-158[3]) and 80ml AD.
31. Fuchsine alkaline aqueous[2]
- Dissolve 1gr Fuchsine (1A-308[3]) by heating in 20ml ethanol and 80ml AD;
- filter after cooling.
32. Fuchsine alkaline alcoholic[2]
- Dissolve 3gr Fuchsine (1A-308[3]) by heating in 80ml ethanol and 20ml AD;
- filter after cooling.
33. Fuchsine acid[1]
- Dissolve 1gr Fuchsine (1B-525[3]) by heating in 99ml AD;
- filter after cooling.
34. Fuchsine ponceau[1] (Trichrome according to Goldner).
- Dissolve 0.2gr Ponceau de Xylidine (1B-207[3]) and 0.6gr fuchsine acid (1B-525[3]) in 300ml AD;
- Add 0.6ml glacial acetic acid (3D-087[3]).
35. Gallocyanin-chromiumalin according to Einarson[1] (DNS, RNS, mast cells, 'Nissl' substance, etc.)
- Dissolve 5gr Chromium alumina (3G-103[3]) in 100ml AD;
- Add 0.15gr Gallocyanin (1A-204[3]) and boil while stirring. The dye, which is difficult to dissolve at room temperature, dissolves into a deep blue colour;
- Leave to rest for 5min (no longer);
- After cooling, filter and top up with AD to 100ml;
- the PH value should be around 1.65.
36. Gallocyanin chromiumalin according to Einarson[2] (DNS, RNS, mast cells 'Nissl' substance, etc.)
- Add 5gr Gallocyanin chromium alumina (1A-458[3]) to 100ml AD and boil while stirring;
- filter after cooling.
37. Gentian Violet-Orange G[2]
- Dissolve 1.5gr Gentian violet-orange G (1F-269[3]) in 100ml ethanol under slight heating;
- filter after cooling.
38. of Gieson colouring solution[1] (Connective tissue, overview colouring)
A, tribal solution;
- 500ml saturated picric acid solution (3E-086[3]);
- Dissolve 0.5gr acid fuchsine (1B-525[3]) in 25ml AD;
- filter the picric acid solution and mix with the 25ml fuchsine;
- this stock solution has an unlimited shelf life.
B, use solution;
- to 100ml stock solution add 1ml glacial acetic acid (3D-087[3]) of 1%.
39. of Gieson colouring solution[2] (Connective tissue, overview colouring)
- Dissolve 1gr Picrofuchsin (1A-488[3]) in 100ml AD by boiling.
40. Haemaluin acid according to Mayer[1] (Core staining for e.g. HE staining)
- Dissolve 1g Haematoxylin (5B-535[3]) in 1000ml AD;
- Add 200gr Sodium iodate NaJO₃ (3G-061[3]) and dissolve;
- Add 50gr Kalialuin (3G-029[3]) and dissolve. The solution turns blue violet;
- Add 50gr Chloralhydrate (3G-102[3]) and 1gr Citric Acid (3G-087[3]) and mix;
- the solution turns red violet.
The solution is ready to use immediately and is stable for a long time in a tightly closed bottle.
41. Haemalinic acid according to Mayer[2] (Core colouring for e.g. HE colouring)
- Dissolve 10gr Haemaluin (1A-528[3]) by heating in 100ml AD;
- filter after cooling.
42. Haemaluin according to Mayer[2] (Core staining for e.g. HE staining)
- Dissolve 5gr Haemaluin (1A-552[3]) by boiling in 100ml AD;
- filter after cooling.
43. Haematoxylin according to Delafield[2] (Core staining for e.g. HE staining)
- Dissolve 2g Haematoxylin (1A-368[3]) by heating in 70ml AD;
- Add 15ml methyl alcohol (3E-158[3]) and 15ml glycerin (3D-086[3]);
- filter after cooling.
44. Haematoxylin according to Ehrlich[1] (Core staining for e.g. HE staining)
- Dissolve 2 g Haematoxylin (5B-535[3]) by heating in 100 ml ethanol 96%;
- Dissolve 3gr Kalialuin (3G-029[3]) by boiling in 100ml AD;
- in the still warm alum solution mix 100ml glycerin (3D-086[3]);
- mix the haematoxylin solution in small portions in the alum solution;
- add 10ml glacial acetic acid (3D-087[3]).
Allow the colour solution to mature in a dark open bottle (cover with a piece of filter paper) for at least 2 weeks. Shake regularly. The solution has a long shelf life.
If you do not want to wait for ripening, you can immediately prepare the dye by adding an oxidizing agent. Add on 1gr haematoxylin 0,2gr sodium iodate NaJO₃ (3G-061[3]).
This haematoxylin gives a very powerful dark blue core colour. Because of the glacial acetic acid, the danger of over colouring is strongly reduced and, also because of the glycerine, the shelf life is increased.
45. Haematoxylin according to Ehrlich[2] (Core staining for e.g. HE staining)
- Dissolve 2g Haematoxylin (1A-508[3]) by boiling in 40ml AD, 20ml ethanol 96% and 40ml glycerin (3D-086[3]);
- after boiling add 5ml glacial acetic acid (3D-087[3]);
- after cooling, filter.
46. Iron haematoxylin according to Heidenhains[1] (Core colouring for e.g. Gieson)
A;
- Dissolve 10gr Iron Alumina (= Ammonium Iron (III) sulphate) (3G-095[3]) in 100ml AD without heating;
B;
- Dissolve 0.5gr Haematoxylin (5B-535[3]) in 10ml ethanol 96% and dilute with 90ml AD;
- allow this solution to mature for 4-5 weeks in an open bottle.
Dilute solution 'B' before use 1:1 with AD. The colour solution can be used several times and will improve even after a longer period of time.
Artificial maturation by adding an oxidizing agent is possible. Add to 0,5gr haematoxylin 98,5mgr sodium iodate NaJO₃ (3G-061[3]).
47. Iodide-potassium iodide[2] (Lugol)
- Dissolve 2gr potassium iodide (3L-005[3]) in 5ml AD;
- Add 1gr iodine (3L-004[3]) which will dissolve in a few minutes;
- supplement with AD to 300ml.
48. Core red aluminium sulphate[1] (Core colouring)
- Dissolve 5gr Aluminium Sulfate (3G-092[3]) in 100ml AD;
- heat the aluminium sulphate solution and dissolve 0.1gr of core red (1A-402[3]);
- filter after cooling.
49. Core red aluminium sulphate[2] (Core colouring)
- Dissolve 5.2gr Core red aluminum sulphate (1A-466[3]) by heating in 95ml AD;
- filter after cooling.
50. Core redrubin[2] (Core colouring)
- Dissolve 0.1gr Kernechtrubin (1A-268[3]) in 100ml AD without heating;
- after dissolving add 1 ml glacial acetic acid (3D-087[3]).
51. Lithium carmine according to Orth[1]
- Dissolve 1g Lithium Carbonate (3G-043[3]) in 100ml AD (provides a saturated solution);
- filter the solution just before use;
- Add 2.5g Carmine (5A-380[3]) to the solution and boil for 5-10min;
- filter after cooling.
To preserve, add 1 thymol (3L-013[3]).
52. Lithium carmine according to Orth[2]
- Dissolve 2.5g Lithium carmine (1A-386[3]) by boiling in 100ml AD;
- after cooling, filter twice (the solution must be clear);
To preserve, add 1 thymol (3L-013[3]).
53. Mallory's trichrome[1]
- see recipe 3 or 4
54. Methylene blue aqueous[2] (core colouring)
- Dissolve 2gr Methylene Blue (1B-429[3]) by heating in 10ml ethanol and 90ml AD;
- filter after cooling.
55. Methylene blue alcoholic[2] (core colouring)
- Dissolve 3gr Methylene Blue (1B-429[3]) by heating in 80ml ethanol and 20ml AD;
- filter after cooling.
56. Methyl green pyronin according to Pappenheim[2] (DNS, green and RNS, red).
- Dissolve 1gr Methylgreen-pyronin (1A-560[3]) by heating in 100ml AD and 5ml ethanol;
- filter after cooling.
Just before staining, mix the dye 1:1 with sodium acetate PH 4.8[4].
57. Sodium acetate buffer according to Walpole[1]
A;
- Dissolve 8.2gr sodium acetate (3G-052[3]) in 1000ml AD.
B;
- Dissolve 5,8ml glacial acetic acid (3D-087[3]) in 1000ml AD.
Mix in ml according to the table below:
PH | Solution A | Solution B |
| 3,6 | 15 | 185 |
| 3,8 | 24 | 176 |
| 4,0 | 36 | 164 |
| 4,2 | 53 | 147 |
| 4,4 | 74 | 126 |
| 4,6 | 98 | 102 |
| 4,8 | 120 | 80 |
| 5,0 | 141 | 59 |
| 5,2 | 158 | 42 |
| 5,4 | 171 | 29 |
| 5,6 | 181 | 19 |
58. Methylviolet aqueous[2]
- Dissolve 2gr Methyl Violet (1B-417[3]) by heating in 100ml AD;
- filter after cooling.
59. Methylviolet alcoholic[2]
- Dissolve 2gr Methyl Violet (1B-415[3]) in 70ml ethanol and 30ml AD;
- filter.
60. Picrine-blue black according to Heidenhain[1] (basal membranes)
- Dissolve 0.25gr Blueblack B (1F-239[3]) in 100ml picric acid (3E-086[3]);
- Add 20ml methyl alcohol (3E-158[3]) and then 80ml AD.
The dye has an unlimited shelf life. For staining 1:1 dilute with AD.
61. Picro-indigo carmine according to Mayer[1]
- Dissolve 0.25gr Indigo carmine (1A-162[3]) in 70ml picric acid (3E-086[3]).
62. Resorcinfuchsin according to Refuses[1] (elastic fibers)
A;
- 0.5gr Fuchsine alkaline (1A-308[3]) and 1gr resorcin (3G-072[3]) by heating dissolve in 50ml AD.
B;
- 2gr iron (III) chloride (3L-003[3]) dissolve 10ml AD.
- Bring solution A to boil;
- add solution B and boil again (5min), stirring regularly;
- filter after cooling;
- collect the filter and filtrate in a flask and add 70 to 100 ml ethanol 96%;
- heat the flask to boiling point, the filtrate will now dissolve;
- after cooling, add 0,7 ml of concentrated hydrochloric acid (3L-012[3]) HCL;
- filter.
The colour solution has a shelf life of several months.
63. Resorcinfuchsine according to Weigert[2] (elastic fibres)
- Dissolve 1gr Resorcin-fuchsine (1A-294[3]) by boiling in 100ml ethanol 96% and 3ml hydrochloric acid (3L-012[3]);
- filter after cooling.
64. Safranin aqueous[2] (core colouring)
- Dissolve 1gr Safranin (1B-463[3]) by boiling in 10ml ethanol 96% and 90ml AD;
- filter after cooling.
65. Safranin alcoholic[2] (core coloration)
- Dissolve 2gr Safranin (1B-477[3]) by heating in 100ml ethanol 96%;
- filter after cooling.
66. Hydrochloric alcohol 3%[2]
- 3ml hydrochloric acid 25% (3L-012[3])mix with 100ml ethanol 70%.
67. Schiff's reagent[1]
- Dissolve 0.5gr Pararosaniline (1B-297[3]) in 15ml 1N HCL;
- Dissolve 0.5gr sodium bisulphite (3G-055[3]) in 85ml AD;
- mix both solutions and allow to mature for 24 hours;
- in case of slight yellowing, add 0.3gr activated carbon and filter;
- If the solution is still red, add a little sodium bisulphite.
68. Oil red[1] (fat colouring when cut from a freezing microtome)
A, strain solution
- Dissolve 0.5gr Oil Red O (1E-242[3]) in 100ml isopropanol 99% (3D-085[3]³).
(Instead of oil red O, fat red 7B (1A-272[3]) can also be used)
B, user solution
- Mix 6 parts of solution A with 4 parts AD;
- Leave to stand for 24 hours;
- filter. If the solution is not used immediately, filter again after some time.
69. Trypane blue[1] (test staining for live and dead cells)
A;
- Dissolve 0.5gr Trypan Blue (1B-187[3]) in 100ml AD.
B;
- Dissolve 0.9gr table salt in 100ml AD.
A and B mix 1:1
Cells that are dead or damaged will turn blue, intact living cells will not be stained.
70. Janus green[1] (staining of mitochondria in living cells)
- Dissolve 0.05gr Janus green (1A-156[3]) in 100ml Ringer's physiological saline solution (see recipe 71).
Color 10-20min in a humid chamber. Mitochondria turn deep blue-green or green. If the staining fails, dilute further.
71. Ringer's physiological saline solution (intended for mammals: J.A. Schraag (1972), Handboek voor het onderwijs in de praktische biologie, publisher: Muusses te Purmerend. ISBN: 9789063080396)
- Dissolve 0.3gr Calcium chloride (3G-101[3]), 0.25gr potassium chloride and 8.5gr sodium chloride (3E-162[3]) in 1000ml AD.
72. Methylene Blue Acid II[1] (for aqueous preparations where paraplast/paraffin has been removed)
A;
- 1gr Methylene Blue (1B-429[3]) by heating dissolve in 100ml AD;
- filter after cooling.
B;
- 1gr Azur II (1A-284[3]) by heating dissolve in 100ml AD;
- filter after cooling.
A and B mix 1:1
Because these dyes rinse out quickly in alcoholic solutions (dehydration) they must be fixed for several hours after colouring with a 5% aqueous ammonium molybdate (3G-005[3]) solution[1] (page 194).
73. Pyronine-Toluidine blue[1] (for aqueous preparations where paraplast/paraffin has been removed)
A;
- 1gr Toluidine blue O (1B-481[3]) by heating dissolve in 100ml AD;
- filter after cooling.
B;
- Dissolve 1gr Pyronin (1B-397[3]) by heating in 100ml AD;
- filter after cooling.
For staining mix 4 parts A with 1 part B.
Since these dyes rinse out quickly in alcoholic solutions (dehydration) they must be fixed for several hours after staining with a 5% aqueous ammonium molybdate (3G-005[3]) solution[1] (page 194).
74. Levanol[1] (myelin staining in nerve tissue)
- Dissolve 2gr Levanol Fast Cyanine 5RN (1E-232[3]) in 90ml methanol (3E-158[3]);
- leave for one night;
- add 10ml glacial acetic acid (3D-087[3]) before use;
- do not filter the saturated solution.
Stain Protocol[1] (page 596):
- fix nerve tissue in formalin and paraplast;
- placing coupes in an aqueous environment;
- 5min coloring in core red aluminum sulfate (1A-466[3]) 0.5%;
- rinsing with AD;
- 5min stain with aqueous phosphorus tungsten acid (3D-092[3]) 1%;
- coils with AD;
- 2-3min coils in methanol (3E-158[3]);
- 5min coloring in levanol (1E-232[3]);
- differentiate into three portions of methanol ice cream vinegar 9:1;
- 3min of methanol;
- 3min xylol (3D-075[3]);
- cover in euparal (3C-239[3]) or malinol (3C-242[3]).
Myelin colours powerful blue (colour contrast is stronger than with luxol fast blue). Coloring is only suitable for the central nervous system. In peripheral nerves, other types of tissue are overdyed.
75. Orange G-eosine Y[1] (staining according to Mann-Dominici)
- 0.2gr Erythrosine Y (1A-336[3]) or 0.5gr eosin Y (1B-427[3]) and 0.6gr Orange G (1B-221[3]) in 100ml AD;
- acidify with 1 drop of glacial acetic acid.
76. Safranin-Gentian Violet-Orange G[2] (Chromosomes, flushing figures and centers according to Flemming)
- Dissolve 0.5gr Safranin (1B-463[3]) in 20ml methanol (3E-158[3]), 80ml AD and 1ml aniline (3D-093[3]).
- Dissolve 1gr Gentian Violet (1B-411[3]) in 20ml methanol (3E-158[3]) and 80ml AD.
- Dissolve 0,5gr Orange G (1B-221[3]) by heating in 90ml AD and 10ml methanol (3E-158[3]);
- filter the Orange G solution after cooling. For staining dilute with AD 1:10.
Tissue must be fixed in Chromium-Osmium-ice vinegar (3D-015[3]) (Flemming). In the case of paraffin blocks that have been stored for years, chromium must still be chromium-plated with Flemming liquid at specimen level for 24 hours to restore the stainability. See Solutions for a recipe for this fixative.
77. Zivernitrate[1] (Silver impregnation according to Servier-Munger, modification of Bielschowsky. Rom. 17, p. 590, par 1.2.5.3.9)
- Dissolve 20gr Silver Nitrate (3F-083[3]) in 50ml AD, then top up with AD to 100ml.
Do not use metal tools and all glassware must be very clean.
78. Silver ammonia solution[1] (Silver impregnation according to Servier-Munger, modification of Bielschowsky Rom. 17, p. 590, par 1.2.5.3.9).
A;
- Dissolve 10g Silver Nitrate (3F-083[3]) in 30ml AD, then make up to 50ml with AD;
- Add Ammonia in drops until brown precipitate has just disappeared (wait for the effect after each drop).
B;
- Dissolve 8gr Sodium Carbonate (Soda) in 30ml AD.
C;
- Add 0.5ml of solution B to solution A.
D;
- add 25dr Ammaniac to solution C and mix well (solution should be clear).
E;
- Add 2ml Formol 37% (3D-084[3]) to 98ml AD.
F, use solution;
- Add 10dr of E under constant shaking to silver ammonia solution (A-D);
- immediately pour the obtained solution over the prepared preparations.
Do not use metal tools and all glassware must be very clean.
Clean glassware containing residues of silver ammonia immediately (dried residues may spontaneously explode[1], p. 509).
Preferences:
[1] Prof. Dr. Peter Böck (1989, 17., neubearbeitete auflage), Romeis Mikroskopische Technik, München. Verleger Urban & Schwarzenberg.
[2] Lösungs- und Herstellungsvorschriften für Mikroskopische Farbstoffe, Waldeck GmbH & Co., 48161 Münster/Germany, Havixbeckerstrasse 62, see www.chroma.de or 'downloads'.
[3] Article number from the catalogue of the company Chroma in Münster (Germany), see www.chroma.de or 'downloads'.