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EMS Technical Data Sheets
Technovit® 9100 Routine staining, Immune Reactions, Enzyme Histochemistry, In-Situ Hybridization
EMS Catalog #14655
The following staining and detection reactions are only important examples of processing hard-cut sections. They also apply to MMA thin sections.
Reagents, antibodies, probes, detection systems are variable.
1. Routine staining
| Counterstaining the sections for immunohistochemistry and enzyme histochemistry | |
| Hematoxylin n. Mayer flowing water in tap water transfer to aqua dest. |
30 sec./RT 10 min/RT |
| Rinse nuclear fast red in aqua dest. | 10 min/RT |
| Methyl green (cleaned; see Romeis) Rinse with aqua dest. |
10-20 min/RT |
| HE staining |
| Same as staining paraffin sections |
| Giemsa staining | |
| Deacrylate sections | |
| Giemsa sol. (Mix fresh!) | 30-40 min/RT |
| Differentiate and dehydrate | |
| Acetone / xylol (95 : 5) | |
| Acetone / xylol (70 : 30) | |
| Acetone / xylol (30 : 70) | |
| Xylol | |
| Masson Goldner staining | |
| Deacrylate sections | |
| Haemalaun (Mayer) | 10 min/RT |
| Tap water | |
| Ponceau acid magenta azophloxin | 45 min/RT |
| 1% Acetic acid | |
| Phosphomolybdic acid / Orange G | 7 min/RT |
| 1% Acetic acid | |
| Light green | 40 min/RT |
| 1% Acetic acid | |
| Ascending alcohol series | |
| Xylol | |
| Cover with Eukitt or similar | |
2. Complete Immune Reaction
| Antibody Incubation | |
| Rinse in 0.01M phosphate buffer (pH 7.4) | |
| Primary antibody Diluted in DAKO Antibody Dilutent |
16 h/4°C or 30-45 min/RT |
| Rinsing buffer | |
| DDAKO EnVision Polymer (GAM/GAR), AP paired |
|
| Detection reaction | |
| Rinsing buffer | |
| Substrate chromogen solution: Fast Red |
15-20 min/RT |
| Counterstaining with hematoxylin to Mayer | |
3. Execution of enzyme histochemistry
| Alkaline and acid phosphatase | |
| Rinse in 0-1M tris buffer (pH 9.4) | 10 min/RT |
| Incubation in the reaction solution 0.1M tris buffer (pH 9.4) Real blue salt Naphthol-AS-BI-phosphate |
2 h/37°C |
| Rinse in aqua dest. | |
| Rinse in 0-1M acetate buffer (pH 5.6) | 10 min/RT |
| Incubation in the reaction solution 0.1M acetate buffer (pH 5.6) Hexanium-pararosaniline solution Naphthol-AS-BI-phosphate |
1 h/37°C |
| Rinse in aqua dest. | |
| Refixate in 4% formalin | 2-3 h/RT |
| Rinse in tap water | |
| Counterstain with methyl green | |
| Naphthol-AS-D chloroacetate esterase (ASD) | |
| Reinse in 0.01M phosphate buffer (pH 7,4) | 5 min/RT |
| Incubation in the reaction solution 0.01M phosphate buffer (pH 6.5) Naphthol-AS-D-Chloroacetate Hexanium-pararosaniline solution |
1 h/RT |
| Rinse in aqua dest. | |
| Counterstain with hematoxylin to Mayer | |
Source of Information
Heraeus Kulzer, 2014