Embedding molds

The 6 mm cylindrical segments can then be embedded by placing them into an appropriate-sized embedding mold, on top of which is placed a prewarmed plastic embedding cassette. Molten wax is then added to the mold and cassette before transferring onto ice and allowed to set. The complete blocks are removed from the mold when sufficiently solidified. 

Place in prewarmed embedding molds with fresh polyethylene glycol and cool on ice at 4°C. 

In many studies it is desirable to gain information from both LM and EM. Methods have been devised for such correlative studies (30-32). Sawada and Esaki s method (33) was designed to address factors that they believed to be important for accurate comparison. These factors are the transparency and thinness of embedded blocks, the flatness of tissue sections, and ease of removal of embedding molds from polymerized Epon blocks. Sawada and Esaki s method is summarized below. 

Fill embedding molds according to the manufacturer s recommendations, transfer the specimen, leave at room temperature to harden and remove molds. 

Embedding molds (Surgipath Disposable Base Molds)... 

Because the particles of raw feed are small, several fractions of the feed can be separately contained in the same thin section, thereby saving much time and money. The making of thin sections, therefore, begins with a plastic embedding mold or other suitable container divided into two chambers with a properly cut business card. The >45-pm insoluble residue and >125-pm sieve fractions of the feed are placed wifh a few drops of epoxy resin in the chambers of the mold and a partial vacuum is drawn for a few minutes. Then the container is filled with the remaining resin, small labels inserted in each chamber, and the preparation allowed to harden on a slide warmer set at approximately 40 to 50 C. 

The equipment shown in Fig. 9 is an example of a suitable vacuum impregnation apparatus. A motor-driven turntable inside the vacuum vessel allows multiple samples to be impregnated simultaneously. A simple pouring mechanism makes it possible to pour the premixed epoxy resin components (cresin and hardener) over the samples or into the embedding molds. 

A variety of molds are available to encapsulate samples for embedding. BEEM type capsules are useful for small pieces of materials that can be placed in the tip which then provides pretrimmed specimen blocks. Gelatin capsules are rounded in shape and thus must be trimmed, but they have the advantage that they can be removed by soaking in water. Other shapes are good for specific sample forms. Flat embedding molds are excellent for films and membranes. 

In the SEM, the samples were first embedded in the epoxy resin Epon-812 (SPI Supplies). The embedded resin was prepared with the formula shown in Table 10.3. A single yam was first placed in an embedding mold made with silicone gel, and then the epoxy resin was added. The mold was initially kept at 30°C for 6 hours, then 60°C for 10 hours and finally 90°C for 8 hours. The oven was slowly cooled down to room temperature before the embedding mold was removed. 

Embedding molds Insect pins, sharpened Surgical carbon-steel razor blade Ultramicrotome, for sectioning Compound microscope Microscope slides... 

To construct the blocks, place a thin layer of Spurr s resin at the bottom of the embedding molds and incubate at 70°C for 3 hours. 

Mount in resin blocks (prepared embedding molds). Carefully place the samples and Spurr s resin in the blocks. If the samples curl up, straightened them by pinning them down into the molds using sharpened insect pins. 

BEEM flat embedding mold (package of 12 Ted Pella 111-22). The type of mold used may vary depending on the microtome model. This size and style works well for a Reichert-Jung microtome. 

PuUed glass micropipette attached to a syringe Embedding molds (see p. 237)... 

Specimen vials Embedding molds Wide-bore plastic pipette Sharpened applicator stick... 

Embedding) Use a large-bore plastic pipette to carefully remove embryos from the vial. The embryos in resin can be placed in a plastic weigh boat. Transfer embryos individually with a sharpened applicator stick to freshly prepared resin in an embedding mold. 

Note There are many different types of embedding molds. Pyramid-tip molds (Ted Pella 10585) are convenient for orienting embryos for longitudinal sectioning. The pyramid tips can later be glued with Superglue to blank blocks polymerized in BEEM (size 00, Ted Pella 130) capsules. 

Embedding molds Embedding oven set at 65°C Specimen vials... 

Embedding) Transfer embryos individually to fresh Epon/Araldite in embedding molds, orient embryos as required, and cure at 65 C for 48 hours to complete polymerization of the Epon/Araldite. 

After sucrose infiltration, dip samples into OCT for at least 5 min and transfer them to an embedding mold containing OCT. Orient samples as desired (reeNote 6) and freeze in dry ice-containing ethanol. Store frozen specimen blocks at -80 °C. See Note 7. 

Assemble the embedding mold, fill the ring mold with agarose solution warmed at 62 °C, allow cooling for about 2 min, and transfer tissue into the agarose solution. Use forceps to orient the tissue in agarose so that it is suspended halfway between the top and bottom of the mold. Incubate at 4 °C until the agarose solidifies. 

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