Preparing Planarian Cells for High-content Fluorescence Microscopy Using RNA in situ Hybridization and Immunocytochemistry

Histone H3 ◽

Nuclear Genome ◽

Quantitative Information ◽

Single Cell Suspension ◽

Proliferating Cells ◽

Rna Transcripts ◽

High-content fluorescence microscopy combines the efficiency of high-throughput techniques with the ability to extract quantitative information from biological systems. The planarian community has developed sensitive and robust assays for whole animals, yet cell based assays, despite their practical aspects, have not been explored to the same extent. Here we describe a modular collection of detailed protocols adapted for fixed planarian cells that enable multiplexed measurements of biomarkers in microwell plates. Methods include the detection of RNA transcripts by RNA fluorescent in situ hybridization combined with tyramide signal amplification using hapten-labeled riboprobes. In addition, immunocytochemical protocols for quantifying proliferating cells by the detection of phosphorylated histone H3 as well as 5-bromo-2'-deoxyuridine incorporation into the nuclear genome are described. The assays are compatible with planarians of virtually any size, as the tissue is disaggregated into a single cell suspension before fixation and staining. By sharing many reagents with established planarian whole mount staining protocols, preparation of samples for high-content microscopy adoption requires little additional investment. Recommendations for successful experimental workflows and common sources of errors are discussed.

Sensitive Whole-Mount Fluorescent In Situ Hybridization in Zebrafish Using Enhanced Tyramide Signal Amplification

Methods in Molecular Biology - Brain Development ◽

10.1007/978-1-62703-655-9_12 ◽

2013 ◽

pp. 175-185 ◽

Cited By ~ 19

Author(s):

Gilbert Lauter ◽

Iris Söll ◽

Giselbert Hauptmann

Keyword(s):

In Situ Hybridization ◽

Signal Amplification ◽

Tyramide Signal Amplification ◽

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

10.17504/protocols.io.bzh5p386 ◽

2021 ◽

Author(s):

Amanda A. G. Ferreira ◽

Bogdan Sieriebriennikov ◽

Hunter Whitbeck

Keyword(s):

Drosophila Melanogaster ◽

In Situ Hybridization ◽

Hybridization Chain Reaction ◽

Chain Reaction ◽

Alexa Fluor ◽

Rna Fish ◽

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).

LNA probes substantially improve the detection of bacterial endosymbionts in whole mount of insects by fluorescent in-situ hybridization

BMC Microbiology ◽

10.1186/1471-2180-12-81 ◽

2012 ◽

Vol 12 (1) ◽

pp. 81 ◽

Cited By ~ 4

Author(s):

Natarajan Priya ◽

Neeti Pandey ◽

Raman Rajagopal

Keyword(s):

In Situ Hybridization ◽

Whole Mount ◽

Bacterial Endosymbionts

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

10.1101/2021.11.01.466817 ◽

2021 ◽

Author(s):

Carlos Guerrero-Hernández ◽

Viraj Doddihal ◽

Frederick G. Mann ◽

Alejandro Sánchez Alvarado

Keyword(s):

In Situ Hybridization ◽

Formic Acid ◽

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.

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

10.1101/2021.08.17.456591 ◽

2021 ◽

Author(s):

Bianca R Maters ◽

Emily Stevenson ◽

Peter D Vize

Keyword(s):

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.

Whole Mount Fluorescent In situ Hybridization of miRNA Combined with Immunofluorescence of Proteins in Intact Islets

Protocol Exchange ◽

10.1038/protex.2018.045 ◽

2018 ◽

Author(s):

Zhiguang Guo ◽

Munir Hossain ◽

Regan Roat ◽

Zhiguang Guo

Keyword(s):

In Situ Hybridization ◽

One FISH, dFISH, Three FISH: Sensitive Methods of Whole-Mount Fluorescent In Situ Hybridization in Freshwater Planarians

In Situ Hybridization Methods - Neuromethods ◽

10.1007/978-1-4939-2303-8_7 ◽

2015 ◽

pp. 127-150 ◽

Cited By ~ 1

Author(s):

David D. R. Brown ◽

Bret J. Pearson

Keyword(s):

In Situ Hybridization ◽

Cytogenetic analysis of Anaecypris hispanica and its relationship with the paternal ancestor of the diploid-polyploid Squalius alburnoides complex

Genome ◽

10.1139/g06-121 ◽

2006 ◽

Vol 49 (12) ◽

pp. 1621-1628 ◽

Cited By ~ 13

Author(s):

Marta Gromicho ◽

Maria Manuela Coelho ◽

Maria Judite Alves ◽

Maria João Collares-Pereira

Keyword(s):

In Situ Hybridization ◽

Cytogenetic Analysis ◽

Nuclear Genome ◽

5S Rdna ◽

Genomic Rearrangements ◽

Endangered Fish ◽

Hybrid Complex ◽

Rdna Sites

The karyotype of the endangered fish Anaecypris hispanica was revisited using advanced cytogenetic techniques to elucidate its putative relationship with the paternal ancestor of the hybrid complex Squalius alburnoides and to clarify some of the recently described cytogenetic patterns of the complex. The results of chromomycin A3 and Ag staining, as well as fluorescent in situ hybridization with 28S and 5S rDNA and the (TTAGGG)n telomeric probes, were compared with the patterns observed in specimens of the all-male nonhybrid lineage of S. alburnoides complex, which is considered to reconstitute the nuclear genome of the probably extinct paternal ancestor. Several cytogenetic features observed in A. hispanica specimens were indeed shared by S. alburnoides nuclear nonhybrid males, supporting the hypothesis of a close evolutionary link between A. hispanica and the paternal ancestor of the complex. The genomic rearrangements involving 28S rDNA sites previously described in the S. alburnoides complex and in its maternal ancestor ( S. pyrenaicus ) were not detected in A. hispanica; they are, therefore, probably due to mechanisms related to hybridization and polyploidy.

In situ microprobe quantitation of inorganic particle burden in paraffin sections of lung tissue

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100107010 ◽

1988 ◽

Vol 46 ◽

pp. 990-991

Author(s):

Jerrold L. Abraham

Keyword(s):

Lung Tissue ◽

Quantitative Information ◽

Particulate Material ◽

Paraffin Sections ◽

Tissue Samples ◽

Qualitative And Quantitative ◽

The Past ◽

Quantitative Analyses ◽

Data Result

Inorganic particulate material of diverse types is present in the ambient and occupational environment, and exposure to such materials is a well recognized cause of some lung disease. To investigate the interaction of inhaled inorganic particulates with the lung it is necessary to obtain quantitative information on the particulate burden of lung tissue in a wide variety of situations. The vast majority of diagnostic and experimental tissue samples (biopsies and autopsies) are fixed with formaldehyde solutions, dehydrated with organic solvents and embedded in paraffin wax. Over the past 16 years, I have attempted to obtain maximal analytical use of such tissue with minimal preparative steps. Unique diagnostic and research data result from both qualitative and quantitative analyses of sections. Most of the data has been related to inhaled inorganic particulates in lungs, but the basic methods are applicable to any tissues. The preparations are primarily designed for SEM use, but they are stable for storage and transport to other laboratories and several other instruments (e.g., for SIMS techniques).