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

Cited by 5 publications

(2 citation statements)

References 14 publications

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“…In short, paraffin-embedded sections were incubated with primary antibodies (DHCR24, #sc-398938, RRID: AB_2832944, 1:100; DHCR24, #sc-390037, RRID: AB_2923495, 1:100; DHCR24, #ab137845, RRID: AB_2923496, 1:100; Tyrosine Hydroxylase (TH), RRID: AB_628422, 1:500; 20αHSD, RRID: AB_2832956, 1:500; CYP2F2, #sc-374540, RRID: AB_10987684, 1:250) followed by appropriate fluorescein-conjugated secondary antibodies. DHCR24 was detected by a biotinylated secondary antibody followed by a fluorescence tyramide [ 39 ]. Fluorescent images were obtained using a Revolve 4 microscope (ECHO).…”

Section: Methodsmentioning

confidence: 99%

DHCR24, a Key Enzyme of Cholesterol Synthesis, Serves as a Marker Gene of the Mouse Adrenal Gland Inner Cortex

Zheng

Kang

Lyu

et al. 2023

IJMS

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Steroid hormones are synthesized through enzymatic reactions using cholesterol as the substrate. In steroidogenic cells, the required cholesterol for steroidogenesis can be obtained from blood circulation or synthesized de novo from acetate. One of the key enzymes that control cholesterol synthesis is 24-dehydrocholesterol reductase (encoded by DHCR24). In humans and rats, DHCR24 is highly expressed in the adrenal gland, especially in the zona fasciculata. We recently reported that DHCR24 was expressed in the mouse adrenal gland’s inner cortex and also found that thyroid hormone treatment significantly upregulated the expression of Dhcr24 in the mouse adrenal gland. In the present study, we showed the cellular expression of DHCR24 in mouse adrenal glands in early postnatal stages. We found that the expression pattern of DHCR24 was similar to the X-zone marker gene 20αHSD in most developmental stages. This finding indicates that most steroidogenic adrenocortical cells in the mouse adrenal gland do not synthesize cholesterol locally. Unlike the 20αHSD-positive X-zone regresses during pregnancy, some DHCR24-positive cells remain present in parous females. Conditional knockout mice showed that the removal of Dhcr24 in steroidogenic cells did not affect the overall development of the adrenal gland or the secretion of corticosterone under acute stress. Whether DHCR24 plays a role in conditions where a continuous high amount of corticosterone production is needed requires further investigation.

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Section: Methodsmentioning

confidence: 99%

DHCR24, a Key Enzyme of Cholesterol Synthesis, Serves as a Marker Gene of the Mouse Adrenal Gland Inner Cortex

Zheng

Kang

Lyu

et al. 2023

IJMS

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“…Then, slides were immersed in boiling sodium citrate solution (10 mM, pH = 6) for 8 min to strip all antibodies used to detect the first antigen. During this procedure, the insoluble fluorophore-tyramide deposition is retained on the tissue ( Toth and Mezey, 2007 ; Francisco-Cruz et al, 2020 ; Lyu et al, 2020 ). Slides were then stained with anti-Sox3 antibodies in a second step, following the regular protocol.…”

Section: Methodsmentioning

confidence: 99%

Otic Neurogenesis in Xenopus laevis: Proliferation, Differentiation, and the Role of Eya1

Almasoudi

Schlosser

2021

Front. Neuroanat.

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Using immunostaining and confocal microscopy, we here provide the first detailed description of otic neurogenesis in Xenopus laevis. We show that the otic vesicle comprises a pseudostratified epithelium with apicobasal polarity (apical enrichment of Par3, aPKC, phosphorylated Myosin light chain, N-cadherin) and interkinetic nuclear migration (apical localization of mitotic, pH3-positive cells). A Sox3-immunopositive neurosensory area in the ventromedial otic vesicle gives rise to neuroblasts, which delaminate through breaches in the basal lamina between stages 26/27 and 39. Delaminated cells congregate to form the vestibulocochlear ganglion, whose peripheral cells continue to proliferate (as judged by EdU incorporation), while central cells differentiate into Islet1/2-immunopositive neurons from stage 29 on and send out neurites at stage 31. The central part of the neurosensory area retains Sox3 but stops proliferating from stage 33, forming the first sensory areas (utricular/saccular maculae). The phosphatase and transcriptional coactivator Eya1 has previously been shown to play a central role for otic neurogenesis but the underlying mechanism is poorly understood. Using an antibody specifically raised against Xenopus Eya1, we characterize the subcellular localization of Eya1 proteins, their levels of expression as well as their distribution in relation to progenitor and neuronal differentiation markers during otic neurogenesis. We show that Eya1 protein localizes to both nuclei and cytoplasm in the otic epithelium, with levels of nuclear Eya1 declining in differentiating (Islet1/2+) vestibulocochlear ganglion neurons and in the developing sensory areas. Morpholino-based knockdown of Eya1 leads to reduction of proliferating, Sox3- and Islet1/2-immunopositive cells, redistribution of cell polarity proteins and loss of N-cadherin suggesting that Eya1 is required for maintenance of epithelial cells with apicobasal polarity, progenitor proliferation and neuronal differentiation during otic neurogenesis.

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

Killinger

Mercado

Choi

et al. 2023

npj Parkinsons Dis.

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Synucleinopathies are neurodegenerative diseases characterized by pathological inclusions called “Lewy pathology” (LP) that consist of aggregated alpha-synuclein predominantly phosphorylated at serine 129 (PSER129). Despite the importance for understanding disease, little is known about the endogenous function of PSER129 or why it accumulates in the diseased brain. Here we conducted several observational studies using a sensitive tyramide signal amplification (TSA) technique to determine PSER129 distribution and function in the non-diseased mammalian brain. In wild-type non-diseased mice, PSER129 was detected in the olfactory bulb (OB) and several brain regions across the neuroaxis (i.e., OB to brainstem). In contrast, PSER129 immunoreactivity was not observed in any brain region of alpha-synuclein knockout mice. We found evidence of PSER129 positive structures in OB mitral cells of non-diseased mice, rats, non-human primates, and healthy humans. Using TSA multiplex fluorescent labeling, we showed that PSER129 positive punctate structures occur within inactive (i.e., c-fos negative) T-box transcription factor 21 (TBX21) positive mitral cells and PSER129 within these cells was spatially associated with PK-resistant alpha-synuclein. Ubiquitin was found in PSER129 mitral cells but was not closely associated with PSER129. Biotinylation by antibody recognition (BAR) identified 125 PSER129-interacting proteins in the OB of healthy mice, which were significantly enriched for presynaptic vesicle trafficking/recycling, SNARE, fatty acid oxidation, oxidative phosphorylation, and RNA binding. TSA multiplex labeling confirmed the physical association of BAR-identified protein Ywhag with PSER129 in the OB and in other regions across the neuroaxis. We conclude that PSER129 accumulates in the mitral cells of the healthy OB as part of alpha-synuclein normal cellular functions. Incidental LP has been reported in the OB, and therefore we speculate that for synucleinopathies, either the disease processes begin locally in OB mitral cells or a systemic disease process is most apparent in the OB because of the natural tendency to accumulate PSER129.

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Microwaving and Fluorophore-Tyramide for Multiplex Immunostaining on Mouse Adrenals − Using Unconjugated Primary Antibodies from the Same Host Species

Cited by 5 publications

References 14 publications

DHCR24, a Key Enzyme of Cholesterol Synthesis, Serves as a Marker Gene of the Mouse Adrenal Gland Inner Cortex

DHCR24, a Key Enzyme of Cholesterol Synthesis, Serves as a Marker Gene of the Mouse Adrenal Gland Inner Cortex

Otic Neurogenesis in Xenopus laevis: Proliferation, Differentiation, and the Role of Eya1

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

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