PEG embedding for immunocytochemistry: application to the analysis of immunoreactivity loss during histological processing.

We have developed a protocol for the production and longterm storage of polyethylene glycol (PEG) sections for immunocytochemistry. Sections obtained by this protocol allow immunolabeling for many different antigens, such as intermediate filaments, macrophage markers, or neurotransmitter enzymes. Standard histological staining can also be performed on these sections. This fixation-embedding system may therefore be of interest for histopathology of rare specimens, as well as for experimental research. Multiple labeling can be performed either on the same section or on consecutive thin sections, thus allowing a more thorough analysis of precious experimental material. We compare the advantages of PEG vs cryostat or vibratome sections. This protocol has been used to study the inactivation of antigenicity by paraffin embedding. We have identified the infiltration by paraffin as the antigenicity inactivating step, not dehydration or high temperature as generally thought.

Three-color immunofluorescence histochemistry allowing triple labeling within a single section.

We describe a procedure for simultaneous immunohistochemical localization of three different neuropeptides, neurotransmitters, or neurotransmitter enzymes within one and the same tissue section and present a number of examples of its application within the brain and periphery. Primary antibodies from three different species were bound to three different neurochemical substances within the same section and were then reacted with three appropriate species-specific antisera conjugated with fluorescein, rhodamine/Texas red, or biotin. The biotinylated secondary antiserum was subsequently reacted with diethylaminocoumarin (DAMC) conjugated to avidin. This combination resulted in green, red, and blue fluorescent labeling of each antigen, respectively. Each fluorescent marker was viewed and photographed discretely using appropriate excitation and suppression filter combinations. The method is well suited for analyzing instances of multiple coexistence at both the level of the cell soma and within terminal regions. More broadly, the feasibility of three-color immunofluorescence histochemistry extends the range with which antigen localization can be used to investigate the morphological bases of relationships and interactions between immunohistochemically characterized neuronal elements.

Pentagastrin on neurotransmitter enzyme activities in the rat gastrointestinal tract

The effects of three doses (5, 100, and 250 micrograms/kg) of pentagastrin on the activities of choline acetyltransferase (ChAT) and acetylcholine esterase (AChE), the neurotransmitter enzymes that synthesize and degrade acetylcholine, and monoamine oxidase (MAO), the degradation enzyme for catecholamines, were investigated. Enzyme activities were assayed in 6 gastrointestinal segments of 21- and 28-day-old and adult rats. All animals were injected intraperitoneally for 7 days with pentagastrin, and the results were compared with age-matched controls receiving saline for 7 days. Plasma and adrenal corticosterone levels were measured. No consistent differences in adrenocortical variables existed between pentagastrin- and saline-treated animals. Similarly, no consistent pentagastrin dose responses of ChAT, AChE, and MAO activities were evident. However, at the highest dose pentagastrin generally produced increases in ChAT activities in 21- and 28-day-old rats, while producing decreases in AChE and MAO activities in 21-day-old rats and increases in 28-day-old animals. There were few significant differences in enzyme activities in adult rats receiving pentagastrin as compared to saline.