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-microm cryostat and 1-3-microm 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-microm cryostat sections, the most striking feature of ISH in the thinner resin (2-3 microm) 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.

The Arctic Cylindrocystis (Zygnematophyceae, Streptophyta) Green Algae are Genetically and Morphologically Diverse and Exhibit Effective Accumulation of Polyphosphate

“…, Zhang and Tung ). In neurons, Nissl bodies stained by toluidine blue have been shown to be rosettes of ribosomes attached to the rough endoplasmic reticulum that may be simultaneously stained by lipophilic fluorescent dyes such as DiOC (Chelvanayagam and Beazley , Singhrao and Nair‐Roberts ).…”

Propidium iodide (PI) stains Nissl bodies and may serve as a quick marker for total neuronal cell count

“…The aim of this experiment was to explore the staining pattern of PI in neurons with large Nissl bodies. After compared the staining pattern of PI with those of fluorescent Nissl stain and hematoxylin in neuronal tissues, we reported a modified staining method that specifically labeled Nissl bodies in neurons using the fluorophore PI (Singhrao and Nair-Roberts, 2010).…”

Informed choices for challenging specimens when choosing methacrylate resin systems for histology

“…Computer-assisted histomorphometry is dependent upon sectioning and staining methods (Dai et al, 2005;Plenk, 2010;Sherif et al, 2006Sherif et al, , 2009 and is important for constructing morphological equivalents of the tissue structures in two-dimensions. X-ray microanalysis of unstained slices of teeth (Arnold and Gaengler, 2007; Arnold et al, 2008), immunohistochemistry (Erben, 1997;Rammelt et al, 2007;Rippstein et al, 2006;Wolf et al, 1992;Yang et al, 2003), immunofluorescence (Rippstein et al, 2006;Singhrao et al, 2009, in situ hybridization (ISH) (Saito et al, 1998;Singhrao and Nair-Roberts, 2010), and fluorescent Tunel assays (Singhrao et al, unpublished data) are all possible using this technique.…”

“…The popularity of these resins lies in their unique ability to polymerize at low temperature and de-acrylate prior to staining. Combination of these two properties enhances the immunohistochemical reactivity and molecular probe penetration (Saito et al, 1998;Singhrao and Nair-Roberts, 2010;Yang et al, 2003) for their retrospective allied histology needs. In addition, Singhrao and Nair-Roberts (2010) demonstrated an enhanced hybridization signal due to better probe penetration from both sides of the ''freefloating'' tissue section.…”

“…The second barrier is to enzymatically digest with proteinase K and the third is to promote section tolerance to high temperature (658C) and lengthy incubation periods facilitating probe penetration and hybridization. The fact that subsequent immunohistochemistry is possible using anti-digoxygenin immunoglobulins and anti-alkaline phosphatase chromogenic detection with nitro blue tetrazolium (NBT) and 5-bromo-4-chloro-3-indolyl phosphate (BCIP) removes all barriers to the ISH technique in sections from tissue embedded in MMA (Technovit 9100 New 1 ) resin (Singhrao and Nair-Roberts, 2010).…”