Page 1
Stain with iron hematoxylin solution acc. to Weigert
10 min
Instructions for use
Hematoxylin solutions A and B acc. to Weigert
Art. No.: X906; X907
To get the working solution (iron hematoxylin solution) mix both solutions A and B at a ratio of 1:1 prior to use. The
working solution is stable for about 8 days at room temperature. It is used when an acidoresistant staining of nuclei is
required, e.g. when working with delimed tissues or for counterstaining during trichrome staining. Pure hematoxylin
stainings will fade under acid conditions after a time and, therefore, are not suitable.
Masson Goldner Trichrome Staining
A successive staining method with three different staining solutions allowing a differentiated visualisation of various
fibers of connective tissue against epithelial and muscle tissue.
Goldner’s stain I: Muscle tissue and cytoplasm red (ponceau), connective tissue also red (acid fuchsine)
Goldner’s stain II: Erythrocytes orange (orange G), connective tissue without colour (phosphotungstic acid)
Goldner’s stain III: Connective tissue green (light green SF)
Principle of staining:
The dyes differ in particle size: Goldner’s stain I contains a fine-particle phase (ponceau), which infiltrates quickly all
structures of tissue, and a coarse-particle phase (acid fuchsine), which works more slowly. At first it stains only the
coarse structures of tissue by masking the fine-particle phase. You have to stop the staining procedure at that point to
avoid an overstaining of the tissue.
During the differentiation step with Goldner’s stain II (phosphotungstic acid) it is important to decolour the connective
tissue as much as possible. Only then it can be stained with Goldner’s stain III (light green).
Additional chemicals required:
Goldner’s stain I (Ponceau-Fuchsine Art. No. 3469)
Goldner’s stain II (Phosphotungstic acid - Orange G, Art. No. 3470)
Goldner’s stain III (Light green SF yellowish, Art. No.3473)
Acetic acid solution 1% (Acetic acid 100%, Art. No.3738)
Usual CLEARing Agents: ROTI®Histol (Art. No. 6640)
ROTICLEAR® (Art. No. A538)
Xylene p.a. (Art. No. 4436)
Appropriate Mounting Media: ROTI®Histokitt (Art. No. 6638), compatible with ROTI®Histol
ROTI®Mount (Art. No. HP68), compatible with ROTICLEAR®
ROTI®Histokitt II (Art. No. T160), compatible with Xylene
Instruction*: For de-waxed, rehydrated specimens.
1.
(Mix solution A+B at a ratio of 1:1) max. 3 min
2. Blue in flowing tap water 10-15 min
3. Stain with Goldner’s stain I 5-
4. Rinse with acetic acid solution 1% 30 sec 9. Dehydrate by ascending alcohol series
5. Stain with Goldner’s stain II
until decolouration of connective tissue
1-3 min (normally sufficient) up to 30 min
Examine by microscope. Specimens may not dry out.
* Acc. to Romeis, Mikroskopische Technik, 18. Auflage, Spektrum Akademischer Verlag (2010)
6. Rinse with acetic acid solution 1% 30 sec
7. Counterstain with Goldner’s stain III 2-5 min
Optionally: Examine by microscope.
8. Wash with acetic acid solution 1% 2-5 min
10. CLEAR with ROTI®Histol, ROTICLEAR® or xylene
11. Mount with appropriate mounting medium
Please note:
Instead of light green solution it is possible to use a
0.1-0.2% solution of anilinblue (Art. No. 4002) for
counterstaining (step 7).
Result: Cell nuclei: dark brown; Cytoplasm, muscle: red; Erythrocytes: orange; Connective tissue: green
SDB-Version: 14.10.2021
Page 2
Van Gieson Trichrome Staining
This staining method allows a differential visualisation of tissue structures in paraffin sections, especially the collagen
connective tissue.
Van Gieson’s solution contains two dyes with very different properties: The fine-particle picric acid
infiltrates quickly all structures of tissue by staining them yellow. The coarse-particle acid fuchsine can
stain only the coarse structures of collagen connective tissue during the short residence time. There
the picric acid is masked. Do not prolong the residence time to avoid the masking of picric acid in other tissue
structures, too (principle of progressive staining).
After staining remove the picric acid as completely as possible from collagen connective tissue for
tissue stained with acid fuchsine tends to fade out when being exposed to acids and bases.
The procedure demands some skill for you have to stop rinsing before the picric acid is also removed from the other
tissue structures (in that case the tissue becomes become reddish).
Additional chemicals required:
Van Gieson’s solution (Art.. No. 3925)
Ethanol denatured: 99.8 % (Art. No. K928), 96 % (T171), 70 % (T913)
HCl-ethanol solution 3 % (Art. No. 6477) – working solution 0.5 %
Usual CLEARing Agents: ROTI®Histol (Art. No. 6640)
ROTICLEAR® (Art. No. A538)
Xylene p.a. (Art. No. 4436)
Appropriate Mounting Media: ROTI®Histokitt (Art. No. 6638), compatible with ROTI®Histol
ROTI®Mount (Art. No. HP68), compatible with ROTICLEAR®
ROTI®Histokitt II (Art. No. T160), compatible with Xylene
Instruction*:
1. De-wax and rehydrate sections (descending alcohol series
finishing off with ethanol 70 %).
2. Stain with iron hematoxylin solution acc. to Weigert
(Mix solution A + B at a ratio of 1:1, solution stable for
8 days at room temp.). 5-10 min
3. Rinse with distilled water
to avoid precipitation of hematein.
4. Examine by microscope: Nuclei should be grey blue,
cytoplasm colorless to max. light grey. If the cytoplasm is
stained too intensive differentiate in HCl-ethanol 0.5%.
2-3 sec
7. Stain with van Gieson‘s solution. 1-3-min
8. Rinse shortly with ethanol 70% and ethanol 96%.
Caution, picric acid is especially soluble in diluted
ethanol!
9. Dehydrate and rinse with ethanol 96%,
finish with 2 x ethanol 100%.
10. CLEAR with CLEARing agent.
11. Mount with appropriate mounting medium.
5. Rinse in tap water to interrupt the differentiation.
6. Blue in flowing tap water. 10 min
* Acc. to Romeis, Mikroskopische Technik, 18. Auflage, Spektrum Akademischer Verlag (2010)
Result:
Cell nuclei: dark blue / dark brown
Collagene fibres: red
Muscle, cytoplasm: yellow
SDB-Version: 14.10.2021
Please note at step 9:
Rinse moderately with highly concentrated ethanol
to remove the picric acid from the connective tissue
(see also above).
Page 3
Elastica van Gieson Staining
Van Gieson trichrome staining is well combinable with elastica staining acc. to Weigert allowing a good overview of
various tissue structures, especially a differentiated visualisation of connective tissue and elastic fibres.
Additional chemicals required:
Van Gieson’s solution (Art. No. 3925)
Ethanol denatured 99.8 % (Art. No. K928), 96 % (T171), 70 % (T913)
Resorcinol fuchsine solution acc. to Weigert (Art. No. X877)
Usual CLEARing Agents: ROTI®Histol (Art. No. 6640)
ROTICLEAR® (Art. No. A538)
Xylene p.a. (Art. No. 4436)
Appropriate Mounting Media: ROTI®Histokitt (Art. No. 6638), compatible with ROTI®Histol
ROTI®Mount (Art. No. HP68), compatible with ROTICLEAR®
ROTI®Histokitt II (Art. No. T160), compatible with Xylene
Instruction*:
1. De-wax and rehydrate sections (descending alcohol
series finishing off with ethanol 80 %).
2. Stain with resorcinol fuchsine solution. 20-30 min 10. Blue in flowing tap water. 10 min
3. Rinse with tap water until stain fades. 11. Stain with van Gieson‘s solution. 1-3-min
4. Rinse with distilled water.
5. Differentiate with ethanol 80%.
6. Rinse with distilled water to interrupt the differentiation. 14. CLEAR with CLEARing agent.
7. Examine by microscope:
Elastic fibres dark violet, background light rose.
8. Stain with iron hematoxylin solution acc. to Weigert
(Mix solution A + B at a ratio of 1:1, solution stable for
8 days at room temp.). 2-3 min
*Acc. to Romeis, Mikroskopische Technik, 18. Auflage, Spektrum Akademischer Verlag (2010)
9. Rinse with distilled water
to avoid precipitation of hematein.
12. Rinse shortly with ethanol 70% and ethanol 96%.
Caution, picric acid is especially soluble in diluted
ethanol!
13. Dehydrate and rinse with ethanol 96%,
finish with 2 x ethanol 100%.
15. Mount with appropriate mounting medium.
Please note at step 13:
Rinse moderately with highly concentrated ethanol to
remove the picric acid from the connective tissue and,
therefore, avoid fading of the staining. Caution: If the
rinsing is too intensive the tissue becomes reddish!
Result:
Elastic fibres: dark violet
Cell nuclei: dark blue / dark brown
Collagene fibres: red
Muscle, cytoplasm: yellow
Please note:
The colour intensity depends on the pre-treatment and the composition of the samples to be stained.
It may initially be necessary to adapt the method to the respective conditions.
l g Danger H225-H319-H336
Hematoxylin solution A acc. to Weigert
X906.1 500ml
Carl Roth GmbH + Co. KG
Schoemperlenstraße 3-5 • 76185 Karlsruhe
P.O. Box 100121 • 76231 Karlsruhe
Phone: +49 (0) 721/ 5606-0
Fax: +49 (0) 721/ 5606-149
info@carlroth.com • www.carlroth.com
The com pany is a l imited partnership with headquarter s in Karl sruhe, reg. court
Mannheim HRA 100055. Roth Chemie Gmb H, with headquarte rs in Karl sruhe, reg.
court M annheim HRB 100428, is the personally liable partner. Managi ng Director:
André H oudelet. Sales tax i dentification number: DE 1 43621073.
sse 06/2021
h Danger H290-H318
Hematoxylin solution B acc. to Weigert
X907.1 500ml
SDB-Version: 14.10.2021
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