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Electron Microscopy Sciences

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 seystems 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

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Technovit® 9100 Methyl Methacrylate