A Method for 3D Immunostaining and Optical Imaging of the Mouse Brain Demonstrated in Neural Progenitor Cells

Abstract

It is important to understand changes in cell distribution that occur as a part of disease progression. This is typically achieved using standard sectioning and immunostaining, however, many structures and cell distribution patterns are not readily appreciated in two-dimensions, including the distribution of neural stem and progenitor cells in the mouse forebrain. Three-dimensional immunostaining in the mouse brain has been hampered by poor penetration. For this reason, we have developed a method that allows for entire hemispheres of the mouse brain to be stained using commercially available antibodies. Brains stained for glial fibrillary acidic protein, doublecortin and nestin were imaged in three-dimensions using optical projection tomography and serial two-photon tomography. This staining method is simple, using a combination of heat, time and specimen preparation procedures readily available, so that it can be easily implemented without the need for specialized equipment, making it accessible to most laboratories.

StereoMate: 3D Stereological Automated Analysis of Biological Structures

“…A recent interest in tissue clearing methods have resulted in the development of several new protocols, all promising to deliver high quality 3D reconstructions of nervous system tissue [1][2][3][4][5][6][7][8][9] .…”

Molecular imaging of biological systems with a clickable dye in the broad 800- to 1,700-nm near-infrared window

“…In vitro molecular imaging of cells and tissues in 2D or 3D space (15)(16)(17) could strongly benefit from the broader spectral imaging window extending into NIR-II, adding more colors and increased multiplexing capability, accompanied by the advantages of increased penetration depth and reduced tissue endogenous autofluorescence levels afforded by molecular NIR-II probes (12,18,19). The increased photon penetration depth allows for visualization of deeper physiological structures, opening the possibility of layer-by-layer fluorescence imaging for 3D molecular imaging using simple one-photon techniques (9,20,21).…”

Antibody Incubation at 37°C Improves Fluorescent Immunolabeling in Free-Floating Thick Tissue Sections

“…However, our preliminary results (data not shown) suggest that the enhancement of antibody penetration into thick tissue sections by antibody incubation at 37C were dependent on intense chemical or physical pretreatment to loosen tissue structures. This could explain the controversial results of previous studies (8,14,15) in which antibody incubation at 37C without tissue loosening were shown to have little or no ability to increase antibody diffusion.…”

“…One possible mechanism for this effect could be that the higher mobility of antibody during incubation at 37C promotes antibody penetration across the section depth (14), and enhancement of interactions between flexible antigen epitopes and highly mobile antibodies at 37C improves immunolabeling sensitivity (15) and specificity. However, our preliminary results (data not shown) suggest that the enhancement of antibody penetration into thick tissue sections by antibody incubation at 37C were dependent on intense chemical or physical pretreatment to loosen tissue structures.…”

“…It has been suggested that an increased antibody incubation temperature (such as 37C, the mammalian body temperature), in contrast to the conventionally used incubation temperatures (4C or 21C), improves fluorescent immunolabeling of thick tissue sections, possibly through enhancement of the penetration (14) or specific binding (15) of the antibody. Moreover, antibody incubation at 37C has been extensively adopted in recently developed protocols for 3-D fluorescent immunolabeling of large tissue samples (such as iDISCO, ScaleS, Clarity, CUBIC, and SWITCH) (37).…”