Technovit® 9100 was specifically developed for the embedding of mineralized tissues as well as soft tissue with an expanded study spectrum in light microscopy. The deplasticized sections are suitable for histological overview staining, enzyme chemistry and immunohistological studies, including in-situ hybridization.
Thin sections for immunohistology can be stuck to glass object holders and deplasticized.
Tissues that cannot be cut are teeth-bearing jaw sections with fillings, crowns and bridges, thick corticalis, implant-bearng (metal or ceramic) jaw or long bones, or brittle, hypermineralized bones.
Polymerization of the hydrophobic Technovit® 9100 occurs by excluding oxygen using a catalyst system made of peroxide and amine. Additional components such as PMMA powder and regulator allow for a controlled polymerization in the cold (in the range of -2 to -20°C, depending on the volume) that guarantees complete dissipation of the polymerization heat.
|Technovit® 9100||1 x 1000 ml Basic Solution
1 x 120g PMMA Powder
8 x 1g Hardener 1
1 x 10 ml Hardener 2
1 x 5 ml Regulator
|Technovit® 9100 Basic Solution||5000 ml|
|Technovit® 9100 PMMA Powder||1000g|
|Technovit® 9100 Hardener 1||100 x 1g|
|Technovit® 9100 Hardener 2||9 x 10 ml|
|Technovit® 9100 Regulator||12 x 5 ml|
|Technovit® 9100 Basic Solution Stabilized||1 x 1000 ml||1|
|Technovit® 9100 PMMA Powder||120g||2|
|Technovit® 9100 Hardener 1||8 bags, each 1g||3|
|Technovit® 9100 Hardener 2||10 ml||4|
|Technovit® 9100 Regulator||5 ml||5|
Fixation is done for 12 to 24 hours in various fixation solutions depending on the size of the tissue and the antigen/enzyme to be detected. Overfixation must always be avoided.
NOTE: Processing may only be done in PE or glass containers!
Dehydration occurs in an ascending alcohol series (dehydration machine) at room temperature. Cavities comprised of white bead polymers that negatively impact cutting and the quality of the section form in insufficiently dehydrated tissue. Xylol is used as an intermedium.
Immersion (pre-infiltration 1-3, infiltration) occurs in 3 phases (in the dehydration machine up to pre-infiltration 2). The specified times and minimum times are based on small, spongy and cortical bone tissue specimens and iliac crest biopsies (the times and volume must be adjusted for larger tissue specimens).
|Dehydration, intermedium and pre-infiltration|
|Dehydration 1||Ethanol||70%||>1 h / RT|
|Dehydration 2||Ethanol||80%||>1 h / RT|
|Dehydration 3||Ethanol||96%||>1 h / RT|
|Dehydration 4||Ethanol||96%||>1 h / RT|
|Dehydration 5||Ethanol||abs.||>1 h / RT|
|Dehydration 6||Ethanol||abs.||>1 h / RT|
|Dehydration 7||Ethanol||abs.||>1 h / RT|
|Intermedium 1||Xylol||>1 h / RT|
|Intermedium 2||Xylol||>1 h / RT|
9100 Basic (stab.)
|>1 h / RT|
(Last phase in machine)
|Technovit® 9100 Basic (stab.) + Hardener 1||>1 h / RT|
|Technovit® 9100 (destab.) + Hardener 1||>1 h / 4°C|
|Technovit® 9100 (destab.) + Hardener 1 + PMMA Powder||>1 h / 4°C
After 5 days, change solution
A standard PMMA granulate can also be used for particularly large specimens (endoprostheses). The amount of required polymerization solution is thereby reduced.
Technovit® 9100 basic solution can be used when stabilized and unstabilized. The application of destabilized basic solution guarantees that the results for all immunohistochemical studies are analagous to the paraffin histology. Fill chromatography column with approx. 50g of Al2O3 (active, alkaline, 90) and slowly flow Technovit® 9100 basic solution (material number 1) through it. A column filling with Al2O3 is able to destabilize 3-4 liters of basic solution. The destabilized solution is portioned into sealable brown glass bottles and stored at +4°C for the ongoing processing (max. 5 days) or kept in storage in aliquots at -15°C to -20°C. Destabilized basic solution can be worked with starting with pre-infiltration 3. When working with destabilized MMA basic solution, a lower amount of peroxide can be used for the infiltration solution and stock solution.
|Making the working solutions|
|Designation||Basic Solution||PMMA Powder||Hardener 1||Hardener 2||Polymerization/
|Processing temperature||Storage and shelf life|
|Pre-infiltration 3||200 ml||1g||Room temp||1/2 year at -20°C|
|Infiltration||ad* 250 ml||20g||1g / 2g**||4°C||1/2 year at -20°C|
|Stock solution A||ad* 500 ml||80g||3g / 4g**||4°C||1/2 year at -20°C|
|Stock solution B||ad* 50 ml||4 ml||2 ml||4°C||1/2 year at -20°C|
*Explanation of "ad": When preparing solutions out of solid substances, the final volume adjustment is made only once all of the substance has dissolved. Please use volumetric flasks.
** When using stablized Technovit® 9100 NEW the greater amount of hardener 1 must be used.
The polymerization mixture is poured into the pre-cooled embedding form. Place the infiltrated tissue into the form, pour the polymerization mixture to the brim and subsequently evacuate. Evacuation is done either in the pre-cooled desiccator at 4°C (light vacuum, e.g. water jet pump or vacuum pump at 200 mbar) or in the freezer with externally connected vacuum pump for approx. 10 minutes. Hermetically seal form!
Polymerization occurs in the range of -2°C to -15°C.
Embedding form 25 mm (10 ml) and the cradle insert: -2°C to 4°C in approx. 24 hours.
Polymerization is complete in approximately 24 hours. The polymerization times depend on the polymerization volume and the temperature. The greater the volume of the embedding form, the lower the temperature must be! Larger specimens must therefore be hardened at lower temperatures. In the process, adhere to the cold capacity of the explosion-protected cooling device used (freezer in the refrigerator, deep freezer, freezer, freezer well, e.g. for paraffin blocks with lid clip.
Reproducible results for various specimen sizes are achieved in a deep freezer with variable temperatures between -2°C and -25°C with temperature consistency of +/- 0.5°C. Do not open the containers during polymerization!
Once the specimens have warmed to room temperature after hardening, use Histoform N to block with Histobloc® and Technovit® 3040. First loosen the bolts and remove the lid and film. The block is tightly clamped in the standard object clamp on the rotation microtome for hard-cut sections.
When using round histo-embedding forms the lid and bottom are removed and the specimen is pushed through. It can then be placed directly in the round sample holder on the rotation microtome for hard-cut sections without being blocked.
Depending on the question, polymers are processed using the hard-cutting or division thin section technique
|Xylol||2 - 3 x 20 min.||RT|
|2-methoxyethyl acetate||1 x 20 min.||RT|
|High-purity acetone||2 x 5 min.||RT|
|High-purity acetone||2 x 2 min.||RT|
|3 x 20 min.||RT|
|Descending alcohol series|
Division with point contact technology and cutting with surface contact or line contact processes with corresponding devices.
|Density = spez. weight g/cm3
|Storage Temperature||Max. 25°C|