Microwaving and Fluorophore-Tyramide for Multiplex Immunostaining on Mouse Adrenals &amp;#8722; Using Unconjugated Primary Antibodies from the Same Host Species

Lyu, Qiongxia; Zheng, Huifei Sophia; Laprocina, Karly; Huang, Chen-Che Jeff

doi:10.3791/60868-v

Cited by 1 publication

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“…IHC was performed as described above with the exception that CF-488, CF-568 or CF-647 tyramide conjugates (Biotium) were substituted for biotinyl tyramide at a final concentration of 1 µg / mL. Multiplex labeling was performed by heating samples 90°C for 30 min in citrate buffer to remove the antibody complex following the tyramide reaction like what has been previously described (59, 60). TSA multiplex labeling of PSER129 and PK-resistant alpha-synuclein was achieved by first labeling PSER129 with CF-568-tyramide.…”

Section: Methodsmentioning

confidence: 99%

Distribution of phosphorylated alpha-synuclein in non-diseased brain implicates olfactory bulb mitral cells in synucleinopathy pathogenesis

Killinger

Brundin

Kordower

et al. 2021

Preprint

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Synucleinopathies including Parkinsons disease and dementia with Lewy bodies are neurodegenerative diseases characterized by the intracellular accumulation of the protein alpha-synuclein called Lewy pathology. Alpha-synuclein within Lewy pathology is aggregated into protease resistant filamentous structures and is predominantly phosphorylated at serine 129 (PSER129). Lewy pathology has been hypothesized to spread throughout the nervous system as the disease progresses. Cross-sectional studies have shown the olfactory bulb and olfactory tract consistently bare LP for common synucleinopathies, making these structures likely starting points for the spreading process, and thus disease. Here we examined the distribution of PSER129 in non-diseased brain. To do this we used a sensitive tyramide signal amplification (TSA) technique to detect low abundance endogenous PSER129 under ideal antibody binding conditions. In wild-type non-diseased mice, PSER129 was detected in the olfactory bulb and several brain regions of the olfactory cortex across the neuroaxis (i.e., olfactory bulb to brain stem). PSER129 was particularly apparent in the mitral cell layer and the outer plexiform layer of the olfactory bulb where it was observed as cytosolic/nuclear puncta or fibers, respectively. PSER129 immunoreactivity in the healthy olfactory bulb was abolished by pretreatment of the tissue with proteinase K, pre-absorption of the primary antibody against the purified PSER129 peptide fragment, or the omission of the PSER129 antibody. Furthermore, PSER129 immunoreactivity was not observed in any brain region of alpha-synuclein knockout mice. Dual labeling for the PSER129 and the mitral cell marker TBX21 showed that PSER129 positive structures of the healthy OB were found in mitral cells. We found evidence of the same PSER129 positive structures in the olfactory bulb of non-diseased rats, non-human primates, healthy humans, but not individuals diagnosed with PD. Results suggest biological pathways responsible for alpha-synuclein phosphorylation are constitutively active in OB mitral cells and alpha-synuclein in these cells may be predisposed to pathological aggregation. Pathological seeds originating in mitral cells may act as a source for alpha-synuclein spread competent assemblies that spreads throughout the brain via fibers of the olfactory tract. Future studies should investigate the normal function of alpha-synuclein in the mitral cells of the olfactory bulb, which may give insight into synucleinopathy disease origins.

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Section: Methodsmentioning

confidence: 99%