A simple histological technique to improve immunostaining when using DNA denaturation for BrdU labelling

Boulanger, Jenna J.; Staines, William; Leblanc, Véronique; Khoo, Eve‐Ling; Liang, Jacky; Messier, Claude

doi:10.1016/j.jneumeth.2015.11.006

Cited by 8 publications

(5 citation statements)

References 18 publications

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“…Every fourth section was processed for eYFP (goat anti-GFP, Abcam ab6673, 1:1000; AlexaFluor 488 donkey anti-goat, Abcam ab150129, 1:500) and then post-fixed in 4% paraformaldehyde overnight before being processed for BrdU immunohistochemistry (Boulanger et al, 2016). Following the eYFP pre-stain and post-fixation, sections were denatured in 1M HCl for 30 min at 45°C, then incubated with the rabbit anti-BrdU (ThermoFisher PA5-32256, 1:1000) primary and AlexaFluor 594 goat anti-rabbit (Jackson ImmunoResearch, 111-585-003, 1:500) secondary antibodies (Boulanger et al, 2016; Wojtowicz and Kee, 2006). Sections were then mounted with Vectashield mounting media with DAPI and viewed for colocalization of the GFP and BrdU immunofluorescent signals.…”

Section: Methodsmentioning

confidence: 99%

Novel long-range inhibitory nNOS-expressing hippocampal cells

Wick

Tetzlaff

Krook-Magnuson

2019

eLife

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The hippocampus, a brain region that is important for spatial navigation and episodic memory, benefits from a rich diversity of neuronal cell-types. Through the use of an intersectional genetic viral vector approach in mice, we report novel hippocampal neurons which we refer to as LINCs, as they are long-range inhibitory neuronal nitric oxide synthase (nNOS)-expressing cells. LINCs project to several extrahippocampal regions including the tenia tecta, diagonal band, and retromammillary nucleus, but also broadly target local CA1 cells. LINCs are thus both interneurons and projection neurons. LINCs display regular spiking non-pyramidal firing patterns, are primarily located in the stratum oriens or pyramidale, have sparsely spiny dendrites, and do not typically express somatostatin, VIP, or the muscarinic acetylcholine receptor M2. We further demonstrate that LINCs can strongly influence hippocampal function and oscillations, including interregional coherence. The identification and characterization of these novel cells advances our basic understanding of both hippocampal circuitry and neuronal diversity.

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Section: Methodsmentioning

confidence: 99%

Novel long-range inhibitory nNOS-expressing hippocampal cells

Wick

Tetzlaff

Krook-Magnuson

2019

eLife

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“…To preserve DCX and other immunostaining during the acid/heat denaturation step required for BrdU labeling, a previously described protocol was used (Boulanger et al, 2016). Immunohistochemistry for DCX and GFP were conducted first.…”

Section: Methodsmentioning

confidence: 99%

Doublecortin in Oligodendrocyte Precursor Cells in the Adult Mouse Brain

Boulanger

Messier

2017

Front. Neurosci.

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Key Points Oligodendrocyte precursor cells express doublecortin, a microtubule-associated protein.Oligodendrocyte precursor cells express doublecortin, but at a lower level of expression than in neuronal precursor.Doublecortin is not associated with a potential immature neuronal phenotype in Oligodendrocyte precursor cells.Oligodendrocyte precursor cells (OPC) are glial cells that differentiate into myelinating oligodendrocytes during embryogenesis and early stages of post-natal life. OPCs continue to divide throughout adulthood and some eventually differentiate into oligodendrocytes in response to demyelinating lesions. There is growing evidence that OPCs are also involved in activity-driven de novo myelination of previously unmyelinated axons and myelin remodeling in adulthood. Considering these roles in the adult brain, OPCs are likely mobile cells that can migrate on some distances before they differentiate into myelinating oligodendrocytes. A number of studies have noted that OPCs express doublecortin (DCX), a microtubule-associated protein expressed in neural precursor cells and in migrating immature neurons. Here we describe the distribution of DCX in OPCs. We found that almost all OPCs express DCX, but the level of expression appears to be much lower than what is found in neural precursor. We found that DCX is downregulated when OPCs start expressing mature oligodendrocyte markers and is absent in myelinating oligodendrocytes. DCX does not appear to signal an immature neuronal phenotype in OPCs in the adult mouse brain. Rather, it could be involved either in cell migration, or as a marker of an immature oligodendroglial cell phenotype.

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“…1 K). However, proliferation of the SMARCA4-deficient and successfully transduced subpopulation could not be analyzed separately since acidic pre-treatment required for BrdU stainings destroys GFP epitopes [ 7 ].…”

Section: Resultsmentioning

confidence: 99%

MYC overexpression and SMARCA4 loss cooperate to drive medulloblastoma formation in mice

Göbel,

Godbole,

Schoof

et al. 2023

acta neuropathol commun

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Group 3 medulloblastoma is one of the most aggressive types of childhood brain tumors. Roughly 30% of cases carry genetic alterations in MYC, SMARCA4, or both genes combined. While overexpression of MYC has previously been shown to drive medulloblastoma formation in mice, the functional significance of SMARCA4 mutations and their suitability as a therapeutic target remain largely unclear. To address this issue, we combined overexpression of MYC with a loss of SMARCA4 in granule cell precursors. Both alterations did not increase proliferation of granule cell precursors in vitro. However, combined MYC overexpression and SMARCA4 loss successfully induced tumor formation in vivo after orthotopic transplantation in recipient mice. Resulting tumors displayed anaplastic histology and exclusively consisted of SMARCA4-negative cells although a mixture of recombined and non-recombined cells was injected. These observations provide first evidence for a tumor-promoting role of a SMARCA4 deficiency in the development of medulloblastoma. In comparing the transcriptome of tumors to the cells of origin and an established Sonic Hedgehog medulloblastoma model, we gathered first hints on deregulated gene expression that could be specifically involved in SMARCA4/MYC driven tumorigenesis. Finally, an integration of RNA sequencing and DNA methylation data of murine tumors with human samples revealed a high resemblance to human Group 3 medulloblastoma on the molecular level. Altogether, the development of SMARCA4-deficient medulloblastomas in mice paves the way to deciphering the role of frequently occurring SMARCA4 alterations in Group 3 medulloblastoma with the perspective to explore targeted therapeutic options.

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A simple histological technique to improve immunostaining when using DNA denaturation for BrdU labelling

Cited by 8 publications

References 18 publications

Novel long-range inhibitory nNOS-expressing hippocampal cells

Novel long-range inhibitory nNOS-expressing hippocampal cells

Doublecortin in Oligodendrocyte Precursor Cells in the Adult Mouse Brain

MYC overexpression and SMARCA4 loss cooperate to drive medulloblastoma formation in mice

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