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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

Here, we present a protocol that uses near-infrared dyes in conjunction with immunohistochemistry and high-resolution scanning to assay proteins in brain regions.

Abstract

Neuroscience is the study of how cells in the brain mediate various functions. Measuring protein expression in neurons and glia is critical for the study of neuroscience as cellular function is determined by the composition and activity of cellular proteins. In this article, we describe how immunocytochemistry can be combined with near-infrared high-resolution scanning to provide a semi-quantitative measure of protein expression in distinct brain regions. This technique can be used for single or double protein expression in the same brain region. Measuring proteins in this fashion can be used to obtain a relative change in protein expression with an experimental manipulation, molecular signature of learning and memory, activity in molecular pathways, and neural activity in multiple brain regions. Using the correct proteins and statistical analysis, functional connectivity among brain regions can be determined as well. Given the ease of implementing immunocytochemistry in a laboratory, using immunocytochemistry with near-infrared high-resolution scanning can expand the ability of the neuroscientist to examine neurobiological processes at a systems level.

Introduction

The study of neuroscience concerns an investigation of how cells in the brain mediate specific functions1. These can be cellular in nature such as how glia cells confer immunity in the central nervous system or can involve experiments that aim to explain how the activity of neurons in the dorsal hippocampus leads to spatial navigation. In a broad sense, cellular function is determined by the proteins that are expressed in a cell and the activity of these proteins2. As a result, measuring the expression and/or activity of proteins in brain cells are critical for the study of neuroscience.

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Protocol

The following protocol was approved by the Institutional Animal Care and Use Committee (IACUC) of the University of Delaware. Male Sprague Dawley rats approximately 55–75 days old were used for this protocol.

1. Brain Extraction and Tissue Preparation

  1. Anesthetize rat with isoflurane in anesthesia induction chamber until rat no longer exhibits a response to foot pinch.
  2. Sacrifice rats via rapid decapitation using a guillotine.
  3. Cut skin on skull posterior to .......

Representative Results

Prior to using high-resolution scanning for immunohistochemistry, one should verify that the protocol works. This can be accomplished using a validation assay where brain sections from the same animal are incubated with primary and secondary antibodies, secondary antibody alone, or neither primary nor secondary antibody. Results for such a validation assay are shown in Figure 2. In this reaction we were detecting the immediate early gene c-Jun in the dorsal h.......

Discussion

The results presented in this article show that near-infrared immunocytochemistry in combination with high-resolution scanning can be used to obtain semi-quantitative measures of protein expression in brain tissue. It can also be used to label two proteins simultaneously in the same brain region. We have previously used near-infrared immunohistochemistry to measure immediate early gene expression in multiple brain regions9,10. Immediate early genes can be used as.......

Acknowledgements

The research in this report was funded by a target grant from the NIGMS (1P20GM103653) awarded to DK.

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Materials

NameCompanyCatalog NumberComments
Brain Extraction
Anesthesia Induction ChamberKent ScientificVetFlo-0530SM
Kleine GuillotineHarvard Apparatus73-1920
Friedman RongeurFine Science Tools16000-14used to remove back of skull
Delicate Dissecting ScissorsFischer Scientific08-951-5used to cut upward along midline of skull
Micro SpatulaFischer Scientific21-401-5used to scoop out brain
Glass Microscope SlidesFischer Scientific12-549-6
Immunohistochemical Reaction
 Triton X-100Used as a mild detergent to permeabilize cells after fixing in Paraformaldehyde, also used as mild detergent in combination with host serum and secondary antibody 
Tween-20Used as a small amount of detergent added to TBS  to procuce TBS-T after coverslipping slides with primary antibody
Licor Odyssey scannerLicor Biotechnology Inc.
Image StudioLicor Biotechnology Inc.

References

  1. Kandel, E. R. . Principles of Neural Science. , (2013).
  2. Byrne, J. H., Roberts, J. L. . From Molecules to Networks: An Introduction to Cellular and Molecular Neuroscience. , (2009).
  3. Salami, A., et al.

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