Characterization of the neurogenic niche in the aging dentate gyrus using iterative immunofluorescence imaging

Cole, John Darby; Castillo, Jacobo Sarabia del; Gut, Gabriele; Gonzalez-Bohorquez, Daniel; Pelkmans, Lucas; Jessberger, Sebastian

doi:10.1101/2021.03.03.433747

Cited by 1 publication

(2 citation statements)

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“…To demonstrate the use of PySeq2500 for automated chemistry and imaging, we have developed an automated protocol for hi-plex staining and imaging of intact tissue sections using iterative indirect immunofluorescence imaging (4i). Unlike other spatial proteomics methods 5,18,19 , 4i enables highly multiplexed protein imaging in cultured cells and tissues using widely available conventional antibodies 4,20 . 4i uses a radical scavenging buffer during imaging to prevent photo-crosslinking of primary antibodies to their target antigen.…”

Section: Introductionmentioning

confidence: 99%

“…4i uses a radical scavenging buffer during imaging to prevent photo-crosslinking of primary antibodies to their target antigen. This allows for gentle antibody elution in a mildly denaturing buffer, preserving sample integrity 4,20 . Through iterative cycles of staining, imaging, and antibody elution, 4i can map up to dozens of proteins in a single sample 4 .…”

Section: Introductionmentioning

confidence: 99%

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PySeq2500: An open source toolkit for repurposing HiSeq 2500 sequencing systems as versatile fluidics and imaging platforms

Pandit

Petrescu

Cuevas

et al. 2021

Preprint

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Fluorescence microscopy is a key method in the life sciences. State of the art -omics methods combine fluorescence microscopy with complex protocols to visualize tens to thousands of features in each of millions of pixels across samples. These -omics methods require precise control of temperature, reagent application, and image acquisition parameters during iterative chemistry and imaging cycles conducted over the course of days or weeks. Automated execution of such methods enables robust and reproducible data generation. However, few commercial solutions exist for temperature controlled, fluidics coupled fluorescence imaging, and implementation of bespoke instrumentation requires specialized engineering expertise. Here we present PySeq2500, an open source Python code base and flow cell design that converts the Illumina HiSeq 2500 instrument into an open platform for programmable applications. Customizable PySeq2500 protocols enable experimental designs involving simultaneous 4-channel image acquisition, temperature control, reagent exchange, stable positioning, and sample integrity over extended experiments. To demonstrate accessible automation of complex, multi-day workflows, we use the PySeq2500 system for unattended execution of iterative indirect immunofluorescence imaging (4i). Our automated 4i method uses off-the-shelf antibodies over multiple cycles of staining, imaging, and antibody elution to build highly multiplexed maps of cell types and pathological features in mouse and postmortem human spinal cord sections. As demonstrated here, PySeq2500 enables non-specialists to develop and implement state of the art fluidics coupled imaging methods in a widely available benchtop system.

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