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Immuno-Bed™ Kit

EMS #14260-00

Introduction

The Immuno-Bed embedding formulation is designed to allow easier penetration of lower molecular weight immunoglobulins through the polymerized matrix for demonstration of antigenic sites. Tissue infiltration, embedding, and sectioning procedures are very similar to JB-4 Embedding Kits.

A semi-soluble media, Immuno-Bed does require dehydration to absolute alcohol as it contains some glycol methacrylate. Immuno-Bed is excellent for routine stains, special stains, and histochemical staining. Clearing agents such as xylene and chloroform are not required. The polymerization of Immuno-Bed is exothermic, which is easily controlled by polymerizing on ice or by using refrigeration at 4°C. Immuno-Bed Embedding Kits must be used under a chemical fume hood. Immuno-Bed is compatible with all routine fixation procedures.

Immunostaining procedures that use lower molecular weight antibodies and chromogens are compatible with Immuno-Bed. For higher molecular weight antibodies and chromogens we recommend Electron Microscopy Sciences’ Osteo-Bed Bone Embedding Kit. The Osteo-Bed formulation is a methyl methacrylate that is well suited for bone or for immunohistochemistry on routine histological specimens.

Immuno-Bed specimens can be sectioned at 0.5 to 3.0 microns or thicker and microtomes designed for plastic sectioning are required. Glass, Ralph, or tungsten carbide knives are recommended for sectioning. Tungsten carbide knives are available from Electron Microscopy Sciences.

Note:

It is recommended that the Embedding Kit be used under a fume hood with appropriate gloves. For additional details, see Warnings and Precautions.

Fixation

Specimens can be fixed in 10% Neutral Buffered Formalin or other routine histological fixative. Poly/LEM is a methanol free formalin based fixative for light and electron microscopy developed by Electron Microscopy Sciences. Routine specimen sizes should be no more than 2.0cm X 2.0cm X 2.0cm with fixation at a minimum of four hours to as long as overnight. Fatty or dense tissues should be fixed overnight. Larger specimens will require fixation overnight or longer depending on the specimen size. Fixation can be at room temperature or 4°C. Cold fixation will extend the time required for the specimen to be penetrated and fixed. Please note that Osteo-Bed or JB-4 are the recommended embedding kits for non-decalcified bone samples.

Dehydration

Dehydration can be completed at room temperature or 4°C. This process can also be done with a routine tissue processor, stopped at the end of the last alcohol step, and removed for infiltration. Polymers cannot be used in routine histology tissue processor, at any time. It will void the warranty and possibly begin to polymerize in the systems thereby blocking the lines. Check with the manufacturer prior to attempting infiltration on any unit.

Infiltration

Infiltration Solution Mixing Procedure:

The following amounts of material are used for one 100ml batch of Infiltration Solution:

Immuno-Bed Solution A (Monomer) 100.00ml
Benzoyl Peroxide, Plasticized (Catalyst) 1.25gm

Carefully weigh 1.25gm of catalyst (benzoyl peroxide, plasticized) and add to 100ml Solution A while stirring on a magnetic stirrer. Mix until dissolved, approximately 10 to 20 minutes. Measurement of the catalyst is critical, as it will control the rate of polymerization of the plastic and the amount of heat generated by the exothermic reaction. This infiltration solution can be store for up to two weeks in a dark cool area or in the refrigerator at 4°C.

Infiltration Procedure:

Infiltration is performed at room temperature or 4°C. Do not expose the samples to heat or direct light during infiltration. The specimens should be placed in two to three changes of Infiltration Solution used is approximately 8 to 10 times that of the volume of the specimen. The time in each change is dependent on the size and density of the specimen and can range from as short as 10 minutes for smaller specimens to as long as 90 minutes for larger specimens. We strongly recommend that the user determine the optimal infiltration time for their situation. When infiltration is complete the tissue generally appears translucent and is most cases will sink to the bottom of the container. To allow complete saturation, infiltration should be done on a slow rotator, hematology shaker table, or inverted several times during the infiltration process.

Embedding

The polymerization process should be under anaerobic conditions with the use of block holders, under light vacuum or in an air-tight container.

Prior to mixing the Embedding Solution collect and prepare the following materials; an ice bath, and the specimens. Do not pre-cool the molds as this may cause condensation and prevent even polymerization of the block face. To prevent polymerization from occurring too fast and possible overheating of the tissue it is recommended that the polymerization process for embedding be slowed by completing it in the refrigerator or in a cold room at 4°C. Note that this may extend the polymerization from several hours to overnight. Larger specimens require greater amounts of embedding solution and this may result in an even higher temperature exothermic reaction. For this reason embedding of larger specimens is performed at 4°C. Also, larger specimens will require longer times for complete polymerization and may have more unpolymerized liquid on top of the block.

Embedding Solution Mixing Procedure:

Make fresh Solution A following the directions in Infiltration Solution and Procedures above. Do not use old or used catalyzed Infiltration Solution for the embedding solutions.

The following amounts of material are used for 25ml of embedding solution:

Infiltration Solution 25.0ml
Immuno-Bed Solution B (Accelerator) 1.0ml (Must be an exact measurement)

Mix 25ml of freshly made Infiltration Solution and 1.0ml of Immuno-Bed Solution B thoroughly and begin embedding immediately. The small Embedding Molds from Electron Microscopy Sciences require approximately 1.5 to 2ml of solution per mold. The Block Holder is essential to exclude oxygen during the polymerization process. If Block Holders are not used, cover the molds with an air tight film or place under vacuum at no more than 15psi, preferably in a cold room at 4°C or a refrigerator. BEEM capsules may be capped for embedding. The blocks should be clear to pale yellow. The top of the block may have a liquid film on it that can be removed by draining for drying the block in a desiccator for several hours to overnight.

Deplasticizing and Staining

Immuno-Bed contains some glycol methacrylate that cannot be removed completely from the sections. Sections are picked up from a waterbath on a clean slide and air-dried. Washing with 100% ethanol for 30 seconds to 2 minutes will soften and remove some of the polymerized matrix. The slides can be rehydrated quickly through a series of descending alcohols to water or buffer prior to staining. Routine histological stains can be performed on these slides or immunohistochemical procedures can be done following the same steps as paraffin embedded specimens. Staining times for Immuno-Bed samples may increase and should be carefully monitored to assure the reaction. Digestion with trypsin or protease K can be done after the first alcohol step to enhance the permeability of the sample for Immunostaining.

Warning

May be harmful if swallowed. Use only under a hood and with appropriate gloves. Components may cause irritation or allergic skin reaction. Avoid inhalation of vapor. Wash thoroughly hands or exposed areas at once, after handling.

Precautions:

Do not heat over an open flame. Avoid electrical or static sparks. Polymerize only in an electric oven meeting all codes for explosion proof operation. Store un-catalyzed resins at room temperature in the original containers.

Storage

Refrigeration of the kit components is not requires. The components should be stored in a cool dark place. Do Not Store in the light or in a heated area as it may cause the monomer to polymerize. The catalyst, plasticized benzoyl peroxide, is an organic peroxide that is shipped dry and does not require special storage. Please note that the catalyst is formulated to remain stable and weigh correctly for this procedure without any adjustments to the amounts recommended. The catalyst should be kept tightly sealed. The catalyst may decompose with age, therefore we recommend carefully monitoring the date received and using the catalyst only with the kit it came in for best results.

Catalyst Disposal Procedure

The catalyst can be destroyed by slowly adding and mixing the catalyst in 4 times or more the volume to weight of 10% sodium hydroxide solution in water. Do not allow material to settle in lumps or stand in layers, mix until dissolved completely. Dispose of this solution, Monomer A and the accelerator with other hazardous wastes in accordance with local, state, and federal regulations.

First Aid

In case of contact, with any component or mixed solution immediately flush area with water for at least 15 minutes. Should either unpolymerized or polymerized material contact the eyes flush with water for at least 15 minutes. If swallowed drink water to excess and call a physician immediately. Never give anything by mouth to someone who is unconscious.

Online Ordering

The Immuno-Bed™ Kit is available online from the EMS Catalog. For ordering or product information, click here.