Technovit® 9100 Methyl Methacrylate Fixing, Dehydrating, Deplasticizing, Routine Staining and Immunohistochemistry

EMS Catalog #14655

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*

*Please see Technovit® 9100 Methyl Methacrylate Application Information for updates to Solution storage, preinfiltration, and embedding and polymerization. Click here.

Working with polymerized tissue preparations

Using a Microtome

• Preparation of heavy-duty microtome sections using a bench-top rotary microtome
• 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

Enzyme Histochemical Staining

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
	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% NaN 3 solution)
• 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 filte

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

2010 Update to Technivot 9100 New

Please see Technovit® 9100 Methyl Methacrylate Application Information for updates to Solution storage, preinfiltration, and embedding and polymerization. Click here.

Online Ordering

Technovit® 9100 Methyl Methacrylate is available online from the EMS Catalog. For ordering or product information, click here.