Questions? 800-523-5874 | [email protected]
- Prepmaster™ Specimen Preparation Robot
- TEM Grids
- TEM Window Grids
- Omniprobe Nanomanipulation Systems
- K-kit Wet "Liquid" TEM Kit
- Specimen Mounts
- SEM Specimen Holders
- Index and Finder SEM Grids
- SEM for Forensics
- SEM Sample Preparation Station Materials
- Cryogenic Personal Protection Equipment
- Cryo Dewars & Flasks
- Cryogenic Grids & Accessories
- Cryogenic Vials & Racks
- Cooling Chambers & Ice Baths
- Prepmaster™ Specimen Preparation Robot
- Laboratory Microwave Ovens
- LYNX II Automated Tissue Processor
- EMS Poly III
- Microtomes
- Tissue Slicers
- Rapid Immersion Freezer
- Heaters & Chillers
- SEM Cooling Stage
- Glow Discharge Systems
- Sputter Coaters & Carbon Coaters
- Stages
- Freeze Dryers
- Critical Point Dryers
- Cryo-SEM Preparation System
- Specimen Transfer Systems
- Decontaminators
- Desiccators
- Centrifuges
- Dry Baths
- Stirrers, Hot Plates
- Vortexers & Magnetic Mixers
- Rotators & Rockers
- Ovens & Incubators
- Vibration Isolation
- Air Sampling
- Vacuum Pumps
EMS Technical Data Sheets
Technovit® 7100 Routine Staining
EMS Catalog #14653
HEMATOXYLIN-EOSIN
Staining process
| 1. Stain the sections in hematoxylin in accordance with Gill* (filtrate the dye solution) | 15 min |
| 2. Blue in tap water | 10 min |
| 3. Rinse in aqua dest. | |
| 4. Counterstain sections with Eosin | 2-5 min |
| 5. Dehydrate through ethanol 96% and 100% | |
| 6. Clarify with xylen and cover in Eukitt |
Result
| Nucleus | blue |
| Basophilic cytoplasm | blue |
| Acidophilic cytoplasm | pink |
| Muscle tissue | pink |
| Connective tissue | pink |
Solutions
| Hematoxylin in accordance with Gill | |
| Hematoxylin (C.I. 75290) | 6 g |
| Sodium iodate | 0.6 g |
| Aluminum sulphate | 52.8 g |
| Aqua dest. | 690 ml |
| Ethylene glycol | 250 ml |
| Glacial acetic acid | 60 ml |
| Eosin | |
| Eosin Y-(alcoholic) C.I. 45380 | 0.5 g |
| Ethanol 96% | 100 ml |
| Glacial acetic acid | 2 drops |
* After staining with hematoxylin (1) the plastic matric can be decolorized with 0.5 ml of HCL (36%) in ethanol 70%: briefly submerse and then quickly process in tap water (2).
PERIOD ACID SCHIFT (PAS)
Staining process
| 1. 0,4% periodic acid | 30 min, 56°C |
| 2. Rinse in tap water | |
| 3. Aqua dest. rinse 3x | |
| 4. Schiff's reagent | 15 min |
| 5. Rinse thoroughly in tap water | |
| 6. Rinse in aqua dest. | |
| 7. Counterstain sections with hematoxylin in accordance with Gill | 10 min |
| 8. Blue in tap water | 10 min |
| 9. Dehydrate, clarify with xylen and cover in Eukitt |
Note: To avoid a specific pink sheen, one can rinse with sulphite water instead of tap water (5), see also Feulgen.
Result
| Nucleus | blue |
| Glycogen | violet / red |
| Basement membranes | violet / red |
| Mucin | violet / red |
Solutions
| Schiff's reagent | |
| Solution 1: Pararosaniline (C.I. 42500 ) 1 N hydrochloric acid |
0.5 g 15 ml |
| Solution 2: Potassium metabisulphite (K2S205) Aqua dest. |
0.5 g 85 ml |
Mix solution 2, solution 1. After 24 hours (in the dark) the light brown solution is decolorized with 200 mg of bone black (approx. 2 min) and subsequently filtrated.
Store the colorless reagent (leucofuchsin) in the refrigerator.
Gill's hematoxylin: see Hematoxylin-Eosin
FEULGEN
Staining process
| 1. Hydrolise in hydrochloric acid 5 N | 20 min ZT |
| 2. Rinse in aqua dest. 3 x | |
| 3. Schiff's reagent | 15 min |
| 4. Sodium hydrogen sulphite 0.5% | 3 x 2 min |
| 5. Rinse thoroughly with tap water | |
| 6. Dehydrate, clarify with xylen and cover in Eukitt |
Result
| DNS | violet / red |
| Other tissue elements | colorless |
Solutions
| Schiff's reagent |
Hydrochloric acid 5 N
Fill up with 42 ml of hydrochloric acid 36% up to 100 ml Sodium.
| NaHSO3 | 0.5 g |
| Aqua dest. | 100 ml |
GIEMSA
Staining process
| 1. Stain sections in the Giemsa solution (20%) (Giemsa Merck: Dilute 1:5 with aqua dest.) |
1.5 hrs ZT |
| 2. Briefly in acetic acid solution: 4 drops to 100 ml of aqua dest. |
2 sec |
| 3. Submerse in alcohol 96% | |
| 4. Submerse in alcohol 96% | |
| 5. Isopropanol | 3 x 2 min |
| 6. Clarify with xylen and cover in Malinol |
Result
| Nucleus | violet |
| Cytoplasm | blue |
| Erythrocytes pink | pink |
PRUSSIAN BLUE REACTION IN ACCORDANCE WITH PERLS
Staining process
| 1. Potassium ferrocyanide First warm up the solution to 60°C and then filter filtrates |
15 min |
| 2. Rinse in aqua dest. | |
| 3. Safranin O. 0,2% | 2-5 min |
| 4. Rinse in acetic acid 1% | |
| 5. Dehydrate, clarify with xylen and cover in Eukitt |
Result
| Nucleus | red |
| Hemosiderin | blue / green |
Solutions
| Potassium ferrocyanide solution | |
| Potassium ferrocyanide | 1 g |
| Aqua dest. | 50 ml |
| Hydrochloric acid 2% | 50 ml |
| Safranin-Solution | |
| Safranin O. (C.I. 50240) | 0.2 g |
| Acetic acid 1% | 100 ml |
PERIODIC ACID METHENAMINE SILVERC (PAMS) ACCORDING TO JONES
Note: It is recommended to stick on the plastic sections with Mayer's albumin.
Staining process
| 1. Periodic acid 1% | 30 min |
| 2. Rinse in aqua dest. 3x | |
| 3. Methenamine silver solution | 60 min, 60°C |
| 4. Rinse in aqua dest., microscopic test. Sections that have been too weakly stained again in 3 |
|
| 5. If the sections refuse to dissolve despite pre-treatment, dry them on a plate at 60°C in accordance with Point 4. | |
| 6. Gold chloride 0,2% | 1-2 min |
| 7. Rinse in aqua dest. | |
| 8. Sodium thiosulphate 2% | 5 min |
| 9. Rinse in tap water | |
| 10. If necessary, counterstain with HE or safranine O |
|
| 11. Dehydrate, clarify with xylen and cover in Eukitt |
Result
| Basement membranes | brown / black |
Solutions
| Methenamine silver tock solution | |
| a) Hexamethylenetetramine 3 % | 100 ml |
| b) Silver nitrate 5% | 5 ml |
| a) and b) can be stored separately | |
| Methenamine silver stain solution | |
| Stock solution | 50 ml |
| Borax 5% | 5 ml |
| Periodic acid: | 1% (Sigma No. P 7875) |
| Gold chloride: | 0.2% |
| Sodium thiosulphate solution: | 2% (Na2S2O3.5H20) |
DETERMINING ENZYME ACTIVITY
Determination of the enzyme activity in tissues that are embedded in 2 hydroxyethyl methacrylate (GMA) - in particular Technovit® 7100.
Freshly removed tissue is fixated in 4% neutral formaldehyde at 4°C for two hours (immersion). If perfusion fixations are made, very brief fixation times can be adhered to and the enzyme activity is better maintained.
Rinsing fluid
0.1 M cacodylate buffer pH 7.4; the material can possibly be left overnight at 4°C.
Dehydration
1. Alcohol 70% acetone 70%, 30 min at 4°C
2. Alcohol 96% acetone 96%, 30 min at 4°C
3. Alcohol 100% acetone 100%, 30 min at 4°C
Pre-infiltration
4. Alcohol 100% Technovit® 7100 1:1, 2 hrs at 4°C
or
Acetone 100% Technovit® 7100 1:1, 2 hrs at 4°C
Infiltration
Technovit® 7100, 12 hrs at 4°C
Polymerization
15 parts Technovit® 7100 (solution A)
1 part Technovit® 7100 hardener II, at 4°C
The tissue can be embedded in Histoforms S or Q, or in the Sorvall embedding system. Because polymerization starts at 4°C, it will occur slower than at room temperature. A polymerization time of 12 hours at 4°C must be adhered to ensure polymerization.
The 2-µ sections are also dried at room temperature on aqua dest. Enzyme actions can be made without removing the plastic matrix.
Note: It is difficult to detect dehydrogenases.
ALKALINE PHOSPHATASE IN ACCORDANCE WITH BURSTONE
Staining process
| 1. Incubate the plastic sections in the incubation medium. Note: In many cases a 2-hour incubation period is sufficient. |
1-3 hrs |
| 2. Rinse in aqua dest. | 2 min |
| 3. Counterstain the sections with nuclear fast red |
5-10 min |
| 4. Rinse in aqua dest. | |
| 5. Air dry | |
| 6. Cover in malinol |
Result
| Nucleus | red |
| Enzyme activity area | blue |
Note: In this reaction the choice of medium used to cover the material is significant because crystals formation may occur in the reaction product.
Solutions
| Buffer solution | |
| 0.2 M tris-(hydroxymethyl)-aminomethane | 2.4 g |
| Aqua dest. | 100 ml |
| Set the pH value to 8.9 with diluted HCL and store the buffer at 4°C. | |
| Incubation medium | |
| Naphtol AS-MX phosphate, disodium salt (Sigma) | 5 g |
| N,N dimethylformamide | 0.25 ml |
| After dissolving, add: | |
| Aqua dest. | 25 ml |
| Buffer solution (pH 8,9) | 25 ml |
| MgSO4.7H2O 10% | 2 drops |
| Fast Blue BB (Sigma) | 30 mg |
Shake well and then filtrate before using.
Note: Always freshly prepare the incubation medium.
ATP-ASE (WACHSTEIN AND MEISEL)
Staining process
| 1. Incubate the plastic sections in the incubation medium (filtrate before using) Note: In many cases a 2-hour incubation period is sufficient. |
1-3 hrs, 37°C |
| 2. Rinse in aqua dest. | 2 min |
| 3. Sodium sulphide solution | 30 sec |
| 4. Rinse in aqua dest. | |
| 5. Counterstain the sections with nuclear fast red | 5-10 min |
| 6. Rinse in aqua dest. | |
| 7. Air dry | |
| 8. Cover with Eukitt or malinol |
Result
| Nucleus | red |
| Enzyme activity area | brown |
Solutions
| 1. Tris maleic acid buffer pH 7.2 solution A Maleic acid |
29 g |
| Tris-(hydroxymethyl)-aminomethane | 30.3 g |
| Aqua dest. | 500 ml |
| Add 2 g of activated carbon, shake for ten minutes and filtrate. Then add 40 ml of the stock solution A, 20 ml 1N NaOH, and fill with aqua dest. up to 100 ml (pH 7.2). | |
| 2. Lead nitrate solution | |
| Lead nitrate | 2 g |
| Aqua dest. | 100 ml |
| 3. Magnesium sulphate solution | |
| MgSO4.7H2O | 1.2 g |
| Aqua dest. | 100 ml |
| Incubation medium | |
| Aqua dest. | 22 ml |
| Disodium adenosine-5-triphosphate (Boehringer, Mannheim) | 25 mg |
| Tris maleic acid buffer pH 7.2 | 20 ml |
| Magnesium sulphate solution | 5 ml |
| Lead nitrate solution (add by drops), heat to 42°C and filtrate | 3 ml |
| Sulphide solution | |
| Sodium sulphide | 2 g |
| Aqua dest. | 100 ml |
| Adjust the pH value to 7.0-7.5 with 1 N of HCL (verify with pH paper). | |
Source of Information
Heraeus Kulzer, 2014