Cold Spring Harbor Laboratory

7 ARTICLES PUBLISHED IN JoVE

Biology

In Situ Hybridization for the Precise Localization of Transcripts in Plants

Marie Javelle¹, Cristina F. Marco¹, Marja Timmermans¹

The in situ hybridization protocol described here allows a direct localization of mRNA and small RNA expression at the cellular level with high sensitivity and specificity. The procedure is optimized for paraffin-embedded plant tissue sections, is applicable to a wide range of plants and tissues, and can be completed within ten days.

Biology

A Novel Bayesian Change-point Algorithm for Genome-wide Analysis of Diverse ChIPseq Data Types

Haipeng Xing¹, Willey Liao^1,2, Yifan Mo^1,2, Michael Q. Zhang^2,3

¹Department of Applied Mathematics & Statistics, Stony Brook University, ²Computational Biology and Bioinformatics, Cold Spring Harbor Laboratory, ³Department of Molecular and Cell Biology, University of Texas at Dallas

Our Bayesian Change Point (BCP) algorithm builds on state-of-the-art advances in modeling change-points via Hidden Markov Models and applies them to chromatin immunoprecipitation sequencing (ChIPseq) data analysis. BCP performs well in both broad and punctate data types, but excels in accurately identifying robust, reproducible islands of diffuse histone enrichment.

Medicine

Live Imaging of Drug Responses in the Tumor Microenvironment in Mouse Models of Breast Cancer

Elizabeth S. Nakasone^1,2, Hanne A. Askautrud^2,3, Mikala Egeblad²

¹Watson School of Biological Sciences, ²Cold Spring Harbor Laboratory, ³Departments of Medical Genetics, University of Oslo and Oslo University Hospital

We describe a method for imaging response to anti-cancer treatment in vivo and at single cell resolution.

Biology

A Strategy to Validate the Role of Callose-mediated Plasmodesmal Gating in the Tropic Response

Ritesh Kumar^*1, Shu Wei Wu^*1, Arya Bagus Boedi Iswanto¹, Dhinesh Kumar¹, Xiao Han², Jae-Yean Kim¹

¹Division of Applied Life Science (BK21 plus program), Plant Molecular Biology and Biotechnology Research Center, Gyeongsang National University, ²Biotechnology Research Institute, Chinese Academy of Agricultural Sciences

This article describes the methods for screening the genes controlling plasmodesmal permeability and hence auxin gradient during tropic response. This includes the measurement of the degree of tropic response in hypocotyl of Arabidopsis thaliana and checking plasmodesmal permeability by 8-hydroxypyrene-1,3,6-trisulfonic acid (HPTS) loading and finally callose level assessment.

Neuroscience

Single-cell RNA Sequencing of Fluorescently Labeled Mouse Neurons Using Manual Sorting and Double In Vitro Transcription with Absolute Counts Sequencing (DIVA-Seq)

Anirban Paul¹, Z. Josh Huang¹

¹Cold Spring Harbor Laboratory

This protocol describes the manual sorting procedure to isolate single fluorescently labeled neurons followed by in vitro transcription-based mRNA amplification for high-depth single-cell RNA sequencing.

Genetics

A New Toolkit for Evaluating Gene Functions using Conditional Cas9 Stabilization

Polona Šafarič Tepeš^1,2, Raffaella Sordella¹

¹Cold Spring Harbor Laboratory, ²Faculty of Pharmacy, University of Ljubljana

Here, we describe a genome-editing tool based on the temporal and conditional stabilization of clustered regularly interspaced short palindromic repeat- (CRISPR-) associated protein 9 (Cas9) under the small molecule, Shield-1. The method can be used for cultured cells and animal models.

Cancer Research

Longitudinal Intravital Imaging Through Clear Silicone Windows

Laura Maiorino^*1,2,3, Margaret Shevik^*1,4,5, José M. Adrover¹, Xiao Han^1,6, Elias Georgas^7,8, John Erby Wilkinson⁹, Harrison Seidner¹, Leonie Foerschner¹, David A. Tuveson¹, Yi-Xian Qin⁸, Mikala Egeblad¹

¹Cold Spring Harbor Laboratory, ²Cold Spring Harbor Laboratory School of Biological Sciences, ³Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, ⁴Medical Scientist Training Program, School of Medicine, Stony Brook University, ⁵Graduate Program in Pharmacology, Stony Brook University, ⁶Graduate Program in Genetics, Stony Brook University, ⁷Graduate Program in Biomedical Engineering, Stony Brook University, ⁸Department of Biomedical Engineering, Stony Brook University, ⁹Department of Pathology, University of Michigan

An approach is here presented for long-term intravital imaging using optically clear, silicone windows that can be glued directly to the tissue/organ of interest and the skin. These windows are cheaper and more versatile than others currently used in the field, and the surgical insertion causes limited inflammation and distress to the animals.