scholarly journals A powerful and versatile new fixation protocol for immunohistology and in situ hybridization that preserves delicate tissues in planaria

2021 ◽  
Author(s):  
Carlos Guerrero-Hernández ◽  
Viraj Doddihal ◽  
Frederick G. Mann ◽  
Alejandro Sánchez Alvarado
Keyword(s):  
In Situ Hybridization ◽  
Formic Acid ◽  
Fluorescent In Situ Hybridization ◽  
Acid Treatment ◽  
Confocal Imaging ◽  
Proteinase K ◽  
Whole Mount ◽  
Fixation Protocol ◽  
Regeneration Blastema

Whole-mount in situ hybridization (WISH) is a powerful and widely used technique to visualize the expression pattern of genes in different biological systems. Here we describe a new protocol for ISH and immunostaining in the planarian Schmidtea mediterranea. The new Nitric Acid/Formic Acid (NAFA) protocol is compatible with both assays and prevents degradation of the epidermis or blastema. Instead of proteinase K digestion, formic acid treatment is used to permeabilize tissues and preserve antigen epitopes. We show that the NAFA protocol successfully permits development of chromogenic and fluorescent signals in situ, while preserving the anatomy of the animal. Further, the immunostaining of different proteins was compatible with the NAFA protocol following fluorescent in situ hybridization. Finally, we demonstrate with high resolution confocal imaging that the regeneration blastema is preserved when using the new method. This new NAFA protocol will be a valuable technique to study the process of wounding response and regeneration.

HCR RNA-FISH protocol for the whole-mount brains of Drosophila and other insects v1

2021 ◽  
Author(s):  
Amanda A. G. Ferreira ◽  
Bogdan Sieriebriennikov ◽  
Hunter Whitbeck
Keyword(s):  
Drosophila Melanogaster ◽  
In Situ Hybridization ◽  
Fluorescent In Situ Hybridization ◽  
Hybridization Chain Reaction ◽  
Chain Reaction ◽  
Alexa Fluor ◽  
Rna Fish ◽  
Whole Mount

This is a protocol to perform RNA fluorescent in situ hybridization (RNA-FISH) using hybridization chain reaction (HCR) on whole-mount samples of the brains of the fly Drosophila melanogaster and other insects, e.g. the jumping ant Harpegnathos saltator. Probes and HCR reagents are purchased from Molecular Instruments. This protocol is loosely based on the "generic sample in solution" protocol published by Molecular Instruments. Our modifications include the description of fixation conditions, counterstaining by Hoechst, and altered washes. Additionally, we use larger concentrations of probes and hairpins following the protocol described by Younger, Herre et al. 2020. We have successfully employed this protocol to stain insect brains with up to 4 different probe sets simultaneously (hairpins conjugated with Alexa Fluor 488, 546, 496, and 647).

scholarly journals smiFISH and embryo segmentation for single-cell multi-gene RNA quantification in arthropods

2020 ◽  
Author(s):  
Llilians Calvo ◽  
Matthew Ronshaugen ◽  
Tom Pettini
Keyword(s):  
In Situ Hybridization ◽  
Single Cell ◽  
Single Molecule ◽  
Fluorescent In Situ Hybridization ◽  
Single Cells ◽  
Fano Factor ◽  
Confocal Imaging ◽  
Cell Segmentation ◽  
Expression Variability

ABSTRACTRecently, advances in fluorescent in-situ hybridization techniques and in imaging technology have enabled visualisation and counting of individual RNA molecules in single cells. This has greatly enhanced the resolution in our understanding of transcriptional processes. Here, we adapt a recently published smiFISH protocol (single-molecule inexpensive fluorescent in-situ hybridization) to whole embryos across a range of arthropod model species, and also to non-embryonic tissues. Using multiple fluorophores with distinct spectra and white light laser confocal imaging, we simultaneously detect and separate single RNAs from up to eight different genes in a whole embryo. We also combine smiFISH with cell membrane immunofluorescence, and present an imaging and analysis pipeline for 3D cell segmentation and single-cell RNA counting in whole blastoderm embryos. Finally, using whole embryo single-cell RNA count data, we propose two alternative single-cell variability measures to the commonly used Fano factor, and compare the capacity of these three measures to address different aspects of single-cell expression variability.

scholarly journals smiFISH and embryo segmentation for single-cell multi-gene RNA quantification in arthropods

2021 ◽  
Vol 4 (1) ◽  
Author(s):  
Llilians Calvo ◽  
Matthew Ronshaugen ◽  
Tom Pettini
Keyword(s):  
In Situ Hybridization ◽  
Single Cell ◽  
Single Molecule ◽  
Fluorescent In Situ Hybridization ◽  
Single Cells ◽  
Fano Factor ◽  
Confocal Imaging ◽  
Cell Segmentation ◽  
Expression Variability

AbstractRecently, advances in fluorescent in-situ hybridization techniques and in imaging technology have enabled visualization and counting of individual RNA molecules in single cells. This has greatly enhanced the resolution in our understanding of transcriptional processes. Here, we adapt a recently published smiFISH protocol (single-molecule inexpensive fluorescent in-situ hybridization) to whole embryos across a range of arthropod model species, and also to non-embryonic tissues. Using multiple fluorophores with distinct spectra and white light laser confocal imaging, we simultaneously detect and separate single RNAs from up to eight different genes in a whole embryo. We also combine smiFISH with cell membrane immunofluorescence, and present an imaging and analysis pipeline for 3D cell segmentation and single-cell RNA counting in whole blastoderm embryos. Finally, using whole embryo single-cell RNA count data, we propose two alternative single-cell variability measures to the commonly used Fano factor, and compare the capacity of these three measures to address different aspects of single-cell expression variability.

scholarly journals Embryonic and aglomerular kidney development in the bay pipefish, Syngnathus leptorhynchus

2021 ◽  
Author(s):  
Bianca R Maters ◽  
Emily Stevenson ◽  
Peter D Vize
Keyword(s):  
In Situ Hybridization ◽  
Fluorescent In Situ Hybridization ◽  
Histological Analysis ◽  
Kidney Development ◽  
Alpha Subunit ◽  
Kidney Tubule ◽  
Sodium Potassium ◽  
Whole Mount ◽  
Visualization Techniques

In this report we describe the embryogenesis of the bay pipefish, Syngnathus leptorhynchus, and the organogenesis of its aglomerular kidney. Early development was analyzed via a series of montages and images documenting embryos collected from the brood pouches of pregnant males. Despite differences in terminal morphology between pipefish and common teleost models such as medaka and zebrafish, the embryogenesis of these highly advanced fishes is very similar to that of other species. One of the unique features of these fishes is their utilization of an aglomerular kidney. Histological analysis revealed a single long, unbranched kidney tubule in late embryos. The development and structure of this organ was further investigated by cloning the sodium potassium ATPase alpha subunit, atp1a, from S. leptorhynchus and developing whole mount fluorescent in situ hybridization protocols for embryos of this species. Fluorescent stereoscopic and confocal visualization techniques were then used to characterize the 3D morphology of aglomerular kidneys in intact embryos. In all embryonic stages characterized, the aglomerular kidney is a single unbranched tube extending from posterior to the head to the cloaca.

1796: Analysis of Chromosomal Aneuploidy in Patients with Oligoasthenoteratozoospermia(OAT) Using Fluorescent in-situ Hybridization

2007 ◽  
Vol 177 (4S) ◽  
pp. 596-597
Author(s):  
Joseph P. Alukal ◽  
Bobby B. Najari ◽  
Wilson Chuang ◽  
Lata Murthy ◽  
Monica Lopez-Perdomo ◽  
...  
Keyword(s):  
In Situ Hybridization ◽  
Fluorescent In Situ Hybridization ◽  
Chromosomal Aneuploidy
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