Technovit® 9100

  Application
   - Using Components
   - Tissue Preparation
   - Microtomy
   - Staining
   - Recipes &
     Reagents

  Product Details
   - Ordering
     Information

 

   

Technovit® 9100 Methyl Methacrylate

Application Information from Heraeus Kulzer

Using the Components

The basis solution Technovit 9100 NEW can be used in stabilized or destabilized form. The use of destabilized Technovit 9100 NEW basis solution guarantees results for all immunohistochemical methods analogous to those using paraffin sections.

Destabilizing the Basic Solution

Fill a chromatography column with 50 g aluminum oxide and allow the Technovit 9100 NEW basis solution (Component No. 1) to flow slowly through it. A column prepared as above is sufficient to destabilizes 3 - 4 liters of basis solution. Store the destabilized basis solution in portions in stoppered brown glass bottles and store either at 4°C for shorter periods or at-15°C to -20°C for longer periods of time.

Preparation of a ready-to-use solution from the Components 1-5 of the Technovit 9100 Kit

Procedure: To prepare the solutions for pre-infiltration, infiltration and the stock solutions, see the descriptions in the user instructions.

Please note the storage temperatures!

Component Number 1 2 3 4 5  
Component Basic Solution PMMA Powder Hardener 1 Hardener 2 Regulator Storage
Pre infiltration 200 ml   1 g     Room temperature
Infiltration ad 250 ml 20 g 1 g / 2 g*     4°C
Stock solution A ad 500 ml 80 g 3 g / 4 g*     4°C
Stock solution B ad 50 ml     4 ml 2 ml 4°C

*When using stabilized Technovit 9100 NEW the increased amounts of Hardener 1 must be used

Preparation of the Polymerization Mixture

Cooled stock solutions A and B should be mixed in the proportion 9 parts A (measuring cylinder) plus 1 part B (pipette) directly before use. This should be performed in a beaker using a glass rod to stir the mixture. The samples are then positioned in containers and completely covered with polymerization mixture, placed in a cooled desiccator and a partial vacuum produced - for example using a water pump - and left to stand at 4°C for ca.. 10 minutes. The resulting blocks are then placed in a sealed container and left to polymerize at between -8°C and -20°C.

Polymerization

The polymerization process takes place at -8°C to -20°C . The samples are then allowed to stand at 4-8°C (Refrigerator) for at least one hour before being allowed slowly to come to room temperature.

The polymerization times are dependent on the volumes of polymerization mixture used and of the constancy of the temperature at which polymerization is carried out. Larger samples should be polymerized at lower temperatures. The cooling capacity of the refrigerator (ice box, deep freezer, freezer-unit with lid) and the volume of polymerization solution should be taken into account - for example with paraffin blocks with lids - when preparing for polymerization. Reproducible results are achieved in the refrigerator by temperatures from ± 0.5°C when Elektronik-Bau temperature regulator is used.

gelatin capsule 0.2 ml between -8°C and -15°C 18 to 24 h
Teflon mold 3 ml capacity between -8°C and -15°C 18 to 24 h
PVC capsule 15 ml capacity between -8°C and -15°C 18 to 24 h
larger PVC molds (200 - 300 ml) between -15°C and -20°C 24 to 48 h
very large PVC molds 500 - 1000 ml) between -20°C and -25°C 48 h

When the prepared samples have been brought to ambient temperature after polymerization is complete, they can be mounted on blocks using Technovit 3040 in order to remove the samples from the Teflon molds.

Preparation of tissues before embedding

Fixing the tissues

The time for fixing is usually between 12 and 24 h and takes place in different solutions depending not only upon the composition of the specimen but also on the antigen or enzyme to be labeled. The following methods of fixation can be used when detecting antigens or enzymes.

  1. 4% neutral buffered formalin (0.1 mol/l phosphate buffer - or 0.02 mol/l phosphate buffer for pelvic biopsies)
  2. 10% buffered formalin (0.1 mol/l phosphate buffer)
  3. Fixation according to Schaffer (Formol/Alcohol).
  4. 1.4% paraldehyde solution at 4-8°C for 24-48 h. (see table for sensitive detection of enzymes such as alkaline phosphatase and for antigens sensitive to denaturation /structural changes.)
Dehydration, Intermediate and Immersion (Pre-infiltration steps 1 -3, Infiltration)

Dehydration is performed in increasing concentrations of alcohol and can be performed automatically in a suitable device at ambient temperature. If dehydration is incomplete, so-called "Lunker-Stellen" containing pearls of white polymer develop, which can negatively influence the cutting of the block as well as the quality of the sections obtained. Xylol is used as intermediate solution.

Immersion (Pre-infiltration steps 1 -3 plus infiltration) takes place in three stages. Pre-infiltration steps 1 and 2 can be performed automatically in a suitable dehydration device. The times given in the table below are for small spongy and cortical bone samples and pelvic biopsies.

For large tissue samples, the times and volumes should be increased proportionally.

Step Solution Concentration Time
Dehydration-1 Ethanol
70%
1 hour
Dehydration-2 Ethanol
80%
1 hour
Dehydration-3 Ethanol
96%
1 hour
Dehydration-4 Ethanol
96%
1 hour
Dehydration-5 Ethanol
abs.
1 hour
Dehydration-6 Ethanol
abs.
1 hour
Dehydration-7 Ethanol
abs.
1 hour
Intermediate-1 Xylene
1 hour
Intermediate-2 Xylene
1 hour
Pre-infiltration 1 Technovit 9100 NEW (Stabilized)
50%
1 hour
Pre-infiltration 2
(final step when automated)
Technovit 9100 NEW (Stabilized) + Hardener-1
1 hour
Pre-infiltration (Refrigerator) Technovit 9100 NEW (Destabilized) + Hardener-1
1 hour
Pre-infiltration
(Refrigerator)
Technovit 9100 NEW
Basic (Destabilized) + Hardener-1 + PMMA Powder
1 - 2 or 3 days
Working with polymerized tissue preparations
Using a Microtome
  • Preparation of heavy-duty microtome sections using a bench-top rotary microtome such as the JB-4 from EBSciences.
  • As above - but for semi-thin sections using a glass or diamond knife. The blocks must be trimmed before cutting.
  • Use of 16 mm hardened metal knife with D-form cutting edge or HKS-Knives. When cutting polymerized Technovit 9100 NEW blocks, 30% ethanol (cutting fluid) must be used.
  • Transfer sections to super frost plus slides, mount with 50% ethanol (mounting fluid) and cover with PVC-foil (Kisol-foil).
  • Remove excess fluid with filter paper. Load the slides into a section-press
Removal of Polymer from the Sections Prior to Staining - All Steps at Ambient Temperature
Xylene 2-3 x 20 min room temperature
2-methoxyethylacetate (2-MEA) 1 x 20 min room temperature
Pure acetone 2 x 5 min room temperature
Aqua Dest 2 x 2 min room temperature
Alternatively: 2-MEA 3 x 20 min room temperature

Routine Staining, Immune Reactions and Enzyme Immunohistochemistry

General Remarks: The following methods for staining of tissues and for detection of signal reactions are given as important examples for processing heavy-duty microtome sections as an introduction to the polymerization system described in this leaflet. They are analogous to those used with methyl methacrylate (MMA) thin sections.

Routine Staining

Counterstaining of Sections for Immuno- and Enzyme-lmmunohistochemistry

  Haematoxylin according to Mayer
Rinse first with tap-water, slowly changing to distilled water
30 sec at room temperature
10 min at room temperature
  Nuclear Fast Red (C.I. 60760)
Rinse under tap water
10 min at room temperature
  Methyl Green
Rinse with distilled water
10 - 20 min at room temperature

Haematoxylin-Eosin

  as for paraffin sections  

Giemsa Staining

  Remove polymer from sections  
  Fresh Giemsa solution 30 - 40 min at room temperature
Differentiate and Dehydrate  
  Acetone / Xylene (95 : 5)  
  Acetone / Xylene (70 : 30)  
  Acetone / Xylene (30 : 70)  
  Xylene  

Masson-Goldner Staining

  Remove polymer from sections  
  Haemalaun according to Mayer 10 min at room temperature
  Rinse under tap water  
  Ponceau-S-Acid Fuchsin-Azophloxin 45 min at room temperature
  1 % acetic acid  
  Phosphomolybdicacid / Orange-G (CI 16230) 7 min at room temperature
  1 % acetic acid  
 

Light Green SF Yellowish (CI 42095)

40 min at room temperature
  1 % acetic acid  
  increasing concentrations of alcohol  
  Xylene  
  Embed in Eukitt (or similar material)  
Performing the Immune Reaction

Antibody Incubation Step

  Rinse the section with 0.01 mol/l phosphate buffer, pH 7.4  
  Primary antibody diluted in DAKO-antibody diluent 16 h/4°C or 30 - 45 min/37°C
  Rinse with buffer (see above)  
  DAKO EnVision polyvalent antibody (goat-anti-mouse/goat-anti-rabbit) coupled to alkaline phosphatase 30 min at room temperature

Visualization Step

  Rinse with buffer  
 

Chromogenic substrate solution: Fast Red TR

15-20 min at room temperature
  Counterstain with haematoxylin according to Mayer  

With Acid- and Alkaline Phosphatase

  Rinse sections with 0.1 mol/l Tris buffer, pH 9.4 10 min at room temperature
  Incubate in substrate solution 0,1 mol/l Tris buffer pH 9.4 Fast Blue Naphthol-AS-BI-phosphate 2 h/37°C
  Rinse with distilled water  
  Rinse in 0.1 mol/l acetate buffer, pH 5.6 10 min at room temperature
  Incubate in substrate solution Hexonium-Pararosanitine solution
Naphthol-AS-BI-phospate
1 h/37°C
  Rinse with distilled water  
  Fix in 40% formalin 2 - 3 h at room temperature
  Rinse with tap water  
  Counterstain with Methyl Green  

With Esterase Reaction using Naphthol-AS-D-chloracetate

 

Rinse sections with 0.01 mol/l phospate buffer, pH 7.4

5 min at room temperature
  Incubate in substrate solution 1 h/RT 0,1 mol/l phosphate buffer, pH 6.5 Naphthol-AS-D-chloracetate Hexonium-Pararosaniline solution 1 at room temperature
  Rinse with distilled water  
  Counterstain with Haematoxylin according to Mayer.  

Recipes and Reagents

Buffers and Stock Solutions

Sodium Acetate Stock Solution - 2 mol/l

74.13 g sodium acetate
5.5 ml glacial acetic acid
Make up to 500 ml with distilled water.

Sodium Acetate Buffer - 0.1 mol/l, pH 5.6

50 ml stock solution (see above)
950 ml distilled water adjust pH to 5.6 with either sodium hydroxide (pH too low) or acetic acid (pH too high)

Phosphate Stock Solution - 1 mol/l

112.5 g disodium hydrogen phosphate
30 g potassium dihydrogen phosphate
Make up to 1 litre with distilled water,

Phosphate Buffer - 0.01 mot/I, pH 7.4

10 ml phosphate stock solution (see above)
980 ml distilled water adjust to pH 7.4 with o-phosphoric acid or sodium hydroxide
Make up to 1 litre with distilled water

0.04 mol/l Phosphate Buffered 10% Sucrose - pH 7.4

40 ml phosphate stock solution (see above)
100 g sucrose
1g sodium azide (e.g. 10 ml 10% NaN3solution)
850 ml distilled water adjust pH to 7.4 (see above) and make up to 1 litre with distilled water.

Tris Stock Solution - 1 mol/l

121.4 g Tris (hydroxymethyt) aminomethane (Tris)
Make up to 1 litre with distilled water.

Tris Buffer - 0.1 mol/l, pH 9.4

100 ml Tris stock solution (see above)
850 ml distilled water
Adjust pH to 9.4 with hydrochloric acid and make up to 1 litre with distilled water.

Stock solutions are best stored in the dark in stoppered brown glass bottles to prevent microbial growth. Diluted buffers can be stored at 4°C, stock solutions at room temperature.

Fixative Solutions

Buffered Formalin Solution (4%)

100 ml 37% formaldehyde (formalin)
4.5 g potassium dihydrogen phosphate
6.5 g disodium hydrogen phosphate
850 ml distilled water.
Adjust the pH to 7.0 with sodium hydroxide or o-phosphoric acid and make up to 1 litre with distilled water.

Paraformaldehyde Stock Solution - 8%

40 g paraformaldehyde
Make up to 500 ml with distilled water

Paraformadehyde Solution - 1.4%

35 ml paraformaldehyde stock solution (see above)
65 ml distilled water
100 ml 0.04 mol/l phosphate buffered 10% sucrose, pH 7.4 (see above)

Reaction Mixtures

Fast Red Solution

3 ml substrate solution
1 Fast Red tablet
120 µl Levamisole
Mix components in a 5 ml stoppered polystyrene or polyethylene test tube. The solution can be then used for approximately 60 min.

Alkaline Phosphatase Substrate / Reaction Mixture

50 ml Tris buffer - 0.1 mol/l, pH 9.4
50 ml Fast Blue Solution
25 mg Naphthol-AS-Bi-phosphate dissolved in 0.5 ml dimethyl sulphoxide (DMSO) / Triton X-100

Acid Phosphatase Substrate / Reaction Mixture

50 ml acetate buffer - 0.1 mol/l, pH 5.6
500 µl Hexonium-Pararosaniline (250 µl Pararosaniline (CI 42500) in 2 mol/l hydrochloric acid + 250 µl 4% sodium nitrite in distilled water - Vortex and allow to react for 5 min before use)
25 mg Naphthol-AS-BI-phosphate in DMSO / Triton X-100 (see above)

Non-Specific Esterase Substrate Reaction Mixture

50 ml phosphate buffer - 0.1 mol/l, pH 6.5
15 mg Naphthot AS-D-chloroacetate in DMSO / Triton X-100 (see above)
250 µl hexonium-pararosaniline (see above)

Staining Solutions

Giemsa Solution

3 ml Giemsa stock solution (Merck)
97 ml distilled water
1 -2 drops of glacial acetic acid.

Light Green

1 g Light Green SF Yellowish
2 ml glacial acetic acid
Make up to 1000 ml with distilled water.

Phosphomolybdic Acid - Orange-G

30 g phosphomolybdic acid
20 g Orange-G
Make up to 500 ml with distilled water.

- add both solutions together
- filtrate

Ponceau-S - Fuchin• Azophloxin

100 ml Masson's solution
20 ml Azophloxin solution
880 ml 0.2 % acetic acid

For Massons solution mix 1 part of Masson's solution A with 2 parts of Massons solution B.

Massons Solution A: 1 g acid fuchsin (fuchsin-S, acid magenta) made up to 100 ml with distilled water
heat to boiling
add 1 ml glacial acetic acid and filter.
Massons Solution B: 2g Xylidine Ponceau (Ponceau 2R - CI 16150) made up to 200 ml with distilled water
heat to boiling
add 2 ml glacial acetic acid and filter.

Azophloxin Solution

0.5 g azophloxin made up to 100 ml with distilled water
and add 2 ml glacial acetic acid

Source of Information

Heraeus Kulzer, 2002

 


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