Antibody Development and Use in Chromogenic and Fluorescent Immunostaining

Dubaissi, Eamon; Panagiotaki, Niki; Papalopulu, Nancy; Vize, Peter D.

doi:10.1007/978-1-61779-992-1_23

Cited by 6 publications

(5 citation statements)

References 10 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Sections were obtained as described in Dubaissi (Dubaissi et al , ) with modifications. Embryos were embedded in 25% fish gelatine for a minimum of 24 h. 18‐μm‐thick sections were collected and transferred onto superfrost glass slides.…”

Section: Methodsmentioning

confidence: 99%

Dynamic properties of noise and Her6 levels are optimized by miR‐9, allowing the decoding of the Her6 oscillator

et al. 2020

Self Cite

View full text Add to dashboard Cite

Noise is prevalent in biology and has been widely quantified using snapshot measurements. This static view obscures our understanding of dynamic noise properties and how these affect gene expression and cell state transitions. Using a CRISPR/Cas9 Zebrafish her6::Venus reporter combined with mathematical and in vivo experimentation, we explore how noise affects the protein dynamics of Her6, a basic helix‐loop‐helix transcriptional repressor. During neurogenesis, Her6 expression transitions from fluctuating to oscillatory at single‐cell level. We identify that absence of miR‐9 input generates high‐frequency noise in Her6 traces, inhibits the transition to oscillatory protein expression and prevents the downregulation of Her6. Together, these impair the upregulation of downstream targets and cells accumulate in a normally transitory state where progenitor and early differentiation markers are co‐expressed. Computational modelling and double smFISH of her6 and the early neurogenesis marker, elavl3, suggest that the change in Her6 dynamics precedes the downregulation in Her6 levels. This sheds light onto the order of events at the moment of cell state transition and how this is influenced by the dynamic properties of noise. Our results suggest that Her/Hes oscillations, facilitated by dynamic noise optimization by miR‐9, endow progenitor cells with the ability to make a cell state transition.

show abstract

Section: Methodsmentioning

confidence: 99%

Dynamic properties of noise and Her6 levels are optimized by miR‐9, allowing the decoding of the Her6 oscillator

et al. 2020

Self Cite

View full text Add to dashboard Cite

show abstract

“…Immunofluorescence was largely carried out as described previously ( Dubaissi and Papalopulu, 2011 ; Dubaissi et al, 2012 ). All primary antibodies for immunofluorescence were used at a dilution of 1:1000.…”

Section: Methodsmentioning

confidence: 99%

A secretory cell type develops alongside multiciliated cells, ionocytes and goblet cells, and provides a protective, anti-infective function in the frog embryonic mucociliary epidermis

et al. 2014

Self Cite

View full text Add to dashboard Cite

The larval epidermis of Xenopus is a bilayered epithelium, which is an excellent model system for the study of the development and function of mucosal and mucociliary epithelia. Goblet cells develop in the outer layer while multiciliated cells and ionocytes sequentially intercalate from the inner to the outer layer. Here, we identify and characterise a fourth cell type, the small secretory cell (SSC). We show that the development of these cells is controlled by the transcription factor Foxa1 and that they intercalate into the outer layer of the epidermis relatively late, at the same time as embryonic hatching. Ultrastructural and molecular characterisation shows that these cells have an abundance of large apical secretory vesicles, which contain highly glycosylated material, positive for binding of the lectin, peanut agglutinin, and an antibody to the carbohydrate epitope, HNK-1. By specifically depleting SSCs, we show that these cells are crucial for protecting the embryo against bacterial infection. Mass spectrometry studies show that SSCs secrete a glycoprotein similar to Otogelin, which may form the structural component of a mucus-like protective layer, over the surface of the embryo, and several potential antimicrobial substances. Our study completes the characterisation of all the epidermal cell types in the early tadpole epidermis and reinforces the suitability of this system for the in vivo study of complex epithelia, including investigation of innate immune defences.

show abstract

“…Transverse sections were obtained as described in Dubaissi et al (2012) with modifications. Embryos were embedded in 25% fish gelatine and 30% sucrose for a minimum of 24 h. We collected 18 µm thickness hindbrain sections and transferred them onto superfrost glass slides.…”

Section: Methodsmentioning

confidence: 99%

Sequential and additive expression of miR-9 precursors control timing of neurogenesis

et al. 2022

Self Cite

View full text Add to dashboard Cite

MicroRNAs (miRs) have an important role in tuning dynamic gene expression. However, the mechanism by which they are quantitatively controlled is unknown. We show that the amount of mature miR-9, a key regulator of neuronal development, increases during zebrafish neurogenesis in a sharp stepwise manner. We characterize the spatiotemporal profile of seven distinct microRNA primary transcripts (pri-mir)-9s that produce the same mature miR-9 and show that they are sequentially expressed during hindbrain neurogenesis. Expression of late-onset pri-mir-9-1 is added on to, rather than replacing, the expression of early onset pri-mir-9-4 and -9-5 in single cells. CRISPR/Cas9 mutation of the late-onset pri-mir-9-1 prevents the developmental increase of mature miR-9, reduces late neuronal differentiation and fails to downregulate Her6 at late stages. Mathematical modelling shows that an adaptive network containing Her6 is insensitive to linear increases in miR-9 but responds to stepwise increases of miR-9. We suggest that a sharp stepwise increase of mature miR-9 is created by sequential and additive temporal activation of distinct loci. This may be a strategy to overcome adaptation and facilitate a transition of Her6 to a new dynamic regime or steady state.

show abstract

Antibody Development and Use in Chromogenic and Fluorescent Immunostaining

Cited by 6 publications

References 10 publications

Dynamic properties of noise and Her6 levels are optimized by miR‐9, allowing the decoding of the Her6 oscillator

Dynamic properties of noise and Her6 levels are optimized by miR‐9, allowing the decoding of the Her6 oscillator

A secretory cell type develops alongside multiciliated cells, ionocytes and goblet cells, and provides a protective, anti-infective function in the frog embryonic mucociliary epidermis

Sequential and additive expression of miR-9 precursors control timing of neurogenesis

Contact Info

Product

Resources

About