Array tomography protocol
Find out how to perform ultra-high resolution array tomography to uncover previously unseen details of brain molecular architecture.
Full array tomography protocol
Tissue fixation, dehydration and embedding
Immunostaining, elution and data analysis
Array tomography is an ultra-high resolution imaging technique originally developed at Stanford University by Kristina D. Micheva and Stephen J. Smith in order to study neural circuits.
Resin-embedded tissue is cut into ordered arrays of ultra-thin serial sections that are mounted onto microscope slides and stained with fluorescent antibodies. The arrays can be eluted and re-stained multiple times in order to reconstruct a three-dimensional image of antigen distribution.
Array tomography offers multiplex, ultra-high resolution volumetric imaging with depth-independent immunofluorescent staining.
1. Tissue fixation, dehydration and embedding
Discover how to fix, dehydrate and embed tissue for use with array tomography. Tissue fixation immobilizes antigens, whilst preserving molecular architecture. Fixed tissue is dehydrated through immersion in increasing concentrations of ethanol and the water replaced with an acrylic resin. The acrylic resin allows ultra-thin sectioning and repeated immunostaining.
2. Preparation of arrays
Learn how to prepare arrays of serial sections from resin-embedded tissue. Coverslips are treated with a subbing solution to adhere serial sections. The embedded tissue is cut into ultra-thin sections, which are transferred onto coverslips and mounted onto microscopy slides for repeated immunostaining.
3. Immunostaining, elution and data analysis
Read about antibody staining of arrays, repeated elution and data analysis. Arrays are blocked and incubated with fluorescent antibodies before being imaged and analyzed. This process can be repeated multiple times to build a three dimensional image of antigen distribution within single tissue specimens.
All protocols are courtesy of Kristina D. Micheva.