Histologists often arrange tissue samples in a very specific orientation prior to paraffin embedding using a process known as pre-embedding. Pre-embedding has traditionally utilized molten agar that is poured over the tissue and which hardens to maintain the proper orientation of the tissue during subsequent embedding procedures. A shortcoming of using agar as the pre-embedding media is that certain tissues shrink during the embedding process, and the agar-based pre-embedding media limits tissue expansion during slide mounting, resulting in difficulties with the tissue sample adhering to the microscope slide. Jones and Calabresi have provided a simple solution to this challenge by using agar mixed with gelatin as a new pre-embedding media. The authors demonstrate that for central nervous system tissues, which tend to shrink during embedding, using agar-gelatin pre-embedding allows these tissues to completely expand when placed on a 42°C water bath prior to slide mounting. The gelatin is thought to melt away at 42°C, providing space for the shrunken tissue to rehydrate and expand. This simple modification to the pre-embedding process will help histologists pre-embed all types of tissues, preserving the needed orientation prior to paraffin embedding without sacrificing proper tissue adherence to a microscope slide. -Page 569
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 (1,2,3,4,5). 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processing (e.g., rodent spinal cord tissue) allow wax to infiltrate between the pre-embedding medium and the tissue (unpublished observation).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.
Bacto Agar (4%, w/v; BD Diagnostic Systems, Sparks, MD, USA) in distilled water at 40°C is mixed with a 5% (w/v) solution of gelatin A (300 Bloom; Electron Microscopy Sciences, Hatfield, PA, USA) 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 and Use Committee).
Larger tissues (pieces of rodent brain that can stand on edge without toppling) are arranged in a mold (e.g., no. 27147-6; Ted Pella, Redding, CA, USA), and the pre-embedding medium is poured over the top of them. The thickness of the resulting block should be such that tissues are held firmly to allow their removal from the mold without tearing (4-6 mm). Once firmly solidified (≥10 min on a cold surface), the medium around the tissue is trimmed with a razor blade. Agar-gelatin tissue blocks are stored at 4°C submerged in 150 mM NaCl in cassettes until ready for dehydration.
For smaller tissues or tissues that might move from their prearranged position after having embedding medium poured over them, a variation of this technique can be helpful (Figure 2). Rodent spinal cord segments, for example, can be pre-embedded to maintain their rostral-caudal order and to keep them standing on end (for cutting in cross-section) during paraffin processing. Agar-gelatin blocks are cast in molds (Figure 2, step 1). Cores of solidified medium are made with a Miltex biopsy punch (Miltex, York, PA, USA), Harris Uni-Core™ (Electron Microscopy Sciences), or other cutting tool at 3 mm diameter for mouse spinal cord and are removed with pointed forceps (Figure 2, step 2). Tissue pieces are placed in the wells that are created, leaning on the well's walls (Figure 2, step 3). Pre-embedding medium is slowly added along the well wall opposite from the tissue to fill in the remaining spaces around the tissue (Figure 2, step 4). Again, pockets of air must be avoided.
The agar-gelatin block containing the tissue is removed from its mold by cutting around the agar-gelatin block's perimeter with a flat tool. The edges should be trimmed to a wedge shape rather than cutting squarely (Figure 2, step 5); the sample will be anchored in the final paraffin block via the larger face of the wedge. The smaller face will be the cutting surface in the final paraffin block (www.palpath.corn/agar.htm). Processing of tissue and cutting of sections are then done using routine procedures. Although gelatin in an aqueous solution melts at 40°–42°C, it survives paraffin infiltration (60°C) and embedding because it is fixed by the preceding alcohol-mediated dehydration.
Cutting sections from agar-gelatin blocks is performed in the same manner as for blocks containing only paraffin. Once sections are cut and rehydrated in a 42°C water bath for mounting onto slides, the gelatin component melts; this leaves the agar soft and flexible, allowing the tissue to expand as required for proper mounting onto slides (Figure 1B).
Agar or gelatin may bind traces of some histological stains. Additionally, even with antigen retrieval methods that use temperatures around 100°C, the agar portion of the embedding media may persist. However, these will not interfere with analysis of the tissue, because the tissue itself is not infiltrated with the pre-embedding medium.
Overall, the use of a pre-embedding medium empowers the investigator to retain information regarding the orientation and location of the tissues that would otherwise be lost after tissue collection. Agar alone may be suitable for pre-embedding tissues in some cases, but tissues that shrink during processing require the added flexibility provided by a mixture of agar and gelatin to allow the re-expansion of the tissue and proper mounting onto slides. Ultimately, pre-embedding procedures allow the investigator to control how tissue, especially multiple pieces of tissue, are arranged and oriented in the final paraffin block.
M.V.J. developed the described procedure and wrote the manuscript. P.A.C. critically reviewed the manuscript. The author would like to thank Miss Rameeza Allie, Miss Kate Mullen, Dr. Cynthia DeBoy, Dr. Helen Fedor, Dr. Ervin Shaw, Miss Melissa Jones, and Dr. Carlos Pardo for helpful advice and for critical reading of the manuscript. This work was funded by the National Multiple Sclerosis Society and the Nancy Davis Center Without Walls.
The authors declare no competing interests.