Embedding tissue dehydration

After the last experiments, the rats are euthanized by injection of an overdose of pentobarbital and then perfused intracardially with a phosphate-buffered 2.0% paraformadehyde - 2.5% glutaraldehyde fixative. Methyl green solution was injected to confirm the location of the catheter after the perfusion. The spinal cord and nerve roots were dissected out and immersed in the same fixative for 4 h. Two specimens (10 mm rostral and caudal to the conus medullaris from each rat were postfixed with cacodylate-buffered 1 % osmium tetroxide dehydrated in a series of graded alcohol solutions, and embedded in epoxy resin. From the embedded tissue, 1-pm transverse sections were obtained and stained with toluidine blue dyes. Sections obtained from 10 mm rostral to the conus (caudal spinal cord) were used for qualitative evaluation. Quantitative analysis of nerve injury was performed using the sections obtained form 10 mm caudal to the conus. Each fascicle present in the cross section was assigned to an injury score 0 to 3. The injury score for each cross section was then calculated as the average score of all fascicles present in the cross section. 

Performing immunohistochemistry on these rehydrated paraffin sections frequently leads to poor results. In contrast, the same antibodies on paraformaldehyde-fixed and cryostat sections will give good results. The issue with formalin-fixed and paraffin-embedded tissue is that the exposure to formalin and dehydration alters the epitopes in the tissue. As a result, formalin-fixed and paraffin-embedded tissues need additional processing methods, known as epitope retrieval or antigen retrieval. Done before immunohistochemistry, epitope retrieval involves heating the sections in buffer with either an acid or base to allow the antibody to recognize the epitope. Also, the exact process of epitope retrieval can be different for individual antibodies. There are numerous papers and books on epitope retrieval and how to apply the method. 

Fixation tissue samples for immuno histochemistry were fixed in 2% paraformaldehyde, 0.25% glutaraldehyde and 3% sucrose buffered with 0.05M phosphate buffer pH 7. After incubation for 2 hours at 25 °C and 63 hours at 5°C the specimens were washed 3 x 20 min. in phosphate buffer pH 7. Dehydration was carried out using series of ethanol washings 50, 70, 80, 96% followed by 3 x in 99% (V2 hr in each). After additional treatment with 2x2 hrs in petroleum ether (shellsol D70k, Q7712) and 2 x 2 hrs in paraffin with 7% beeswax, the samples were embedded in paraffin. Cross sections of 12.5 /im were made on a Supercut 2050 Reichart Jung pyramitome. 

This chapter offers some select, recent developments regarding fixation, dehydration, and embedding. In addition, some tried-and-true procedures are described for the localizations of cellular and tissue chemicals in stems and roots of young Zea mays seedlings. Also provided are more recently developed fluorochromes for DN A and RNA localizations (18,19). 

Each specimen was dehydrated, infiltrated and embedded in Technovit based methylmethacrylate. One section was cut and around in preparation for scanning electron microscopy (SEM). In each case, three overview photos were necessary and four high magnification fields (40X) were photographed and digitized. These fields were later analyzed for volume fraction of soft tissue, bone... 

In most cases, fixation may be carried out at room temperature. Duration of formalin fixation depends on the nature and the size of the specimen, and may vary from 15 min to 24 h. Longer fixation may be associated with a partial loss of the antigenicity of the component of interest. After formalin fixation, tissue samples are washed in three changes of the buffered saline (PBS) from 15 min to 2 h, but not longer than 24 hours on the whole, since the formaldehyde fixation is partially reversible. After washing in PBS, specimens may be either snap-frozen in liquid nitrogen for subsequent cryosectioning, or dehydrated and embedded in paraffin or synthetic resin. 

Tissue processing tissue specimen (0.5 1.0 mm3) are fixed in 4% buffered formalin for 30 60 min, post-fixed in 1% osmium tetroxide in cacodylate or phosphate buffer, pH 7.2 7.4, stained en bloc for 30 min with 2% aqueous uranyl acetate, then dehydrated in ethanol and embedded in epoxy or acrylic resin. 

Tissue Preparation for Electron Microscopy. Tissues were fixed in 2% paraformaldehyde, 2.5% glutaraldehyde in phosphate buffer (0.1 M, pH 7.4) and 0.02% picric acid. They were then dehydrated in glycol methacrylate monomer and embedded in glycol methacrylate (GMA) (24). 

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