Whole-mount Immunostaining
Whole-mount Immunostaining - Related Videos
08:42
Immunostaining Phospho-epitopes in Ciliated Organs of Whole Mount Zebrafish Embryos
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Cited by 3 •
2016
Techniques are described to immunostain phospho-epitopes in whole zebrafish embryos and then conduct two-color fluorescent confocal localization in cellular structures as small as primary cilia. The techniques for fixing and imaging can define the location and kinetics of the appearance or activation of specific proteins.
09:01
Immunostaining of Whole-Mount Retinas with the CLARITY Tissue Clearing Method
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Cited by 4 •
2021
Here we present a protocol to adapt the CLARITY method of the brain tissues for whole-mount retinas to improve the quality of standard immunohistochemical staining and high-resolution imaging of retinal neurons and their subcellular structures.
05:52
Whole-Mount Immunostaining and Automatic Counting of Mouse Retinal Ganglion Cells
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Cited by 1 •
2024
This protocol describes the procedure for isolating the whole-mount mouse retina and performing immunostaining to label all retinal ganglion cells (RGCs). The process is followed by imaging and automatically counting RGCs using AI-based software, providing a simple, fast, and accurate method for quantifying RGCs in the entire mouse retina.
08:37
Immunostaining of Whole-Mount Drosophila Testes for 3D Confocal Analysis of Large Spermatocytes
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Cited by 6 •
2020
We present here an improved protocol for the whole-mount preparation and immunostaining of Drosophila testes, suitable for confocal microscopy and also allowing reproducible and reliable labeling. We illustrate this protocol by 3D representation and quantification of colocalization experiments on the large S4-S5 spermatocytes.
03:39
Immunostaining of Cells Encapsulated in a 3D Hydrogel System
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2025
This video demonstrates a technique for immunostaining cells encapsulated in a 3D hydrogel. The hydrogel culture containing neural progenitor cells is fixed, permeabilized, and treated with a blocking solution to prevent non-specific immunostaining. A primary antibody cocktail is added to bind specific proteins, followed by fluorescently labeled secondary antibodies and a nuclear stain. The sample is then mounted on a slide and observed under a confocal microscope.
FAQs
Permeabilization is crucial because antibodies must move into the intact specimen to reach targets distributed through its volume. Fixation prepares the embryo, organ, or tissue for staining while maintaining the specimen as a connected structure. Together, these steps determine whether labeling can represent protein localization across the whole structure rather than only at its surface.
The primary antibody provides target recognition by binding the protein of interest. A labeled secondary antibody then makes that binding event visible through fluorescence or another detection mode. This arrangement separates target recognition from signal generation, allowing the detected label to report where the selected protein is located within the intact developmental structure.
Optical sectioning reveals expression patterns at different depths within a labeled specimen, supporting a three-dimensional view of protein distribution. Instead of interpreting only an external surface, researchers can examine how signals occupy the structure as a whole. This is particularly useful when developmental organization depends on the position of proteins across multiple tissue layers.
Keeping the embryo, organ, or tissue intact preserves the spatial relationships among labeled regions across the entire structure. Thin sections provide views of selected planes, whereas whole-mount analysis can show broader patterns of protein localization and tissue organization. This wider context helps researchers recognize defects in morphogenesis or differentiation that may not be apparent in isolated sections.
A typical workflow begins by fixing the intact specimen and permeabilizing it so antibodies can access internal targets. The specimen is then exposed to a primary antibody against the protein of interest, followed by a labeled secondary antibody for visualization. Optical sectioning can subsequently be used to examine the resulting expression pattern in three dimensions.
The method is especially valuable when researchers need to compare protein localization across developmental stages or connect molecular signals with tissue organization. It can also support studies of gene function by showing where a relevant protein appears within an intact structure. Because it preserves whole-structure context, the approach helps identify developmental defects associated with altered differentiation or morphogenesis.