Agar-gelatin for embedding tissues prior to paraffin processing.

Benchmarks Fixed tissues often need to be arranged in a particular configuration , order, or orientation prior to processing for paraffin embedding. Although it is possible to arrange tissues after infiltration (during the final stages of paraffin embedding), it can be more convenient and practical to do this at the time of dissection or tissue collection. Agar has been the classical medium used for this purpose (1–5). Typically, tissues are arranged or oriented as desired, and molten agar is poured over the top of the tissues. The agar solidifies around the fixed tissue (pre-embedding) and is then subjected to routine dehydration and paraffin infiltration procedures, followed by finally embedding the agar-encased tissue in paraffin wax. However, tissues that tend to shrink during processing (e.g., rodent spinal cord tissue) allow wax to infiltrate between the pre-embedding medium and the tissue (unpublished observation). Once sections are laid on a 42°C water bath prior to mounting onto microscopic slides, they rehydrate and expand more extensively than the agar and the layer of wax encasing them. As a result, some regions of the tissues do not lay flat on the slide and are subsequently either torn away or poorly stained during histological procedures (Figure 1A). Gelatin, on the other hand, is also not suitable, on its own, for pre-embedding, since its shape tends to warp, and it becomes very hard after processing for paraffin embedding (unpublished observations). We took advantage of the texture of agar and the low melting point of gelatin to make a pre-embedding matrix that would avoid the problems of each individual medium. distilled water at 40°C is mixed with a 5% (w/v) solution of gelatin A (300 Bloom; in distilled water at a 1:1 ratio to generate a slightly viscous 2% agar:2.5% gelatin solution. The temperature of this medium is used at approximately 40°C to avoid exposing tissues to excessive heat. This medium can be stored at 4°C and remelted at least twice without altering its useful properties. Medium should not be used when it becomes very viscous or too cool, since the increased viscosity prevents the medium from entering small holes and spaces around the tissue, and avoiding these air pockets is crucial to both retaining tissue during processing and proper sectioning later. Perfused and 4% paraformaldehyde-fixed rodent central nervous system (CNS) tissues were used here as an example (using procedures approved by the Johns Hopkins University Animal Care …