In situ hybridization and immunofluorescence on resin-embedded tissue to identify the components of Nissl substance

Abstract

It is not clear whether the Nissl substance is present at the axon hillock. To clarify this gap in knowledge, we conducted in situ hybridization (ISH) on mouse brain tissue using 30-lm cryostat and 1-3-lm acrylic resin sections. Cryostat and rehydrated resin sections were exposed to digoxygenin-labeled glutamic acid decarboxylase 1 sense and antisense riboprobes. Consecutive sections from tissue embedded in resin were subjected to the ribosomal protein L26 primary antibody to determine the distribution of the ribo/polysomes. ISH results from the antisense riboprobe in both cryostat and resin-embedded tissue sections demonstrated an abundance of message in the neurons from the substantia nigra pars reticulate. In addition, the resin sections demonstrated hybridization signal in the axon hillock of some neurons. Immunofluorescence labeling of consecutive sections using an antibody to the most abundant ribosomal protein L26 confirmed their distribution in the cell body and the axon hillock of similar neurons. Compared with the 30-lm cryostat sections, the most striking feature of ISH in the thinner resin (2-3 lm) sections was that there was a phenomenal improvement in the overall clarity and spatial resolution. Reexamination of the axon hillock when continuous with the cell body in cryostat sections revealed that the same message was also present, except it was overlooked initially because of overlapping cell populations in thick tissue slices. As ribosomes are a component of Nissl substance, we propose that the axon hillock, like other parts of the neuron, does contain Nissl substance. Microsc. Res. Tech. 73:555-559, 2010.