Neuroscience

The mammalian neocortex, the outer layer of the cerebrum and most recently evolved brain region, is characterized by its unique areal and laminar organization. Distinct cortical layers and areas can be identified by the protein expression of graded transcription factors and molecular determinants that define the identity of different projection neurons. Thus, specific detection and visualization of protein expression is crucial for assessing the identity of neocortical neurons and, more broadly, for understanding early and late developmental mechanisms and function of this complex system. Several immunostaining/immunofluorescence methods exist to detect protein expression. Published protocols vary with regard to subtle details, which may impact the final outcome of the immunofluorescence. Here, we provide a detailed protocol, suitable for both thin cryostat sections and thick vibratome sections, which has successfully worked for a wide range of antibodies directed against key molecular players of neocortical development. Ranging from early technical steps of brains collection down to image analysis and statistics, we include every detail concerning sample inclusion and sectioning, slide storage and optimal antibody dilutions aimed at reducing non-specific background. Routinely used in the lab, our background-optimized immunostaining protocol allows efficient detection of area- and layer- specific molecular determinants of distinct neocortical projection neurons.

Graphic abstract


Workflow chart for the optimized immunostaining protocol of mouse brain sections. A. A flow chart for different steps of the optimized immunostaining protocol on both thin cryostat and thick vibratome sections. B. Example for immunostaining against Satb2 and Ctip2 on a thin coronal section (20 μm) at the level of the somatosensory cortex. The first column to the left shows the binning system where 6 bins can be overlaid on the image. On the bottom, an example of counting analysis showing the percentage of marker-positive cells normalized to the total number of DAPI or Hoechst-positive cells. C. Example for immunostaining against Satb2 and Ctip2 on a GFP+ thick vibratome section (200 μm). Images are taken at low magnification (10x, left) and high magnification (40x, right). The graph shows a counting of the percentage of Ctip2-positive neurons normalized to the total number of GFP-electroporated neurons on high-magnification images. Images on B and C are modified from Harb et al. (2016).

Oligodendrocytes generate distinct patterns of myelination throughout the CNS. Variations in myelination along axons may enable neurons to fine-tune conduction velocities and alter signal synchronisation. Here we outline a staining protocol permitting the assessment of the number and length of myelin sheaths formed by oligodendrocyte in the mouse grey matter. This protocol enables the investigation of myelination without the need for reporter mice or technically challenging protocols, aiding the investigation of factors influencing myelin production in the brain.

In the developing and mature central nervous system (CNS) the ventricular lumen is lined by the neuroepithelium and ependymal, respectively. These ventricular epithelia perform important functions related to the development, morphogenesis and physiology of the brain. In the mature CNS, ependyma constitutes a barrier between brain parenchyma and cerebro- spinal fluid (CSF). The most prominent feature of the apical surface of ependymal cells is the presence of multiple motile cilia that extend towards the ventricular lumen. The beating of cilia ensures the circulation of the CSF and its impairment leads to hydrocephalus. For an effective CSF flow, ciliary beating must be coordinated at the level of individual cells and at the tissue level. This coordination is achieved through the precise organization of cilia positioning within the plane of the ependyma. Two major features have been described regarding the planar organization of cilia in ependymal cells (Mirzadeh et al., 2010) and both have a cellular and tissular aspect (Boutin et al., 2014). The first one, rotational polarity, refers to the orientation of ciliary beating. At the cellular level, all cilia beat in the same direction (Figure 1B, black arrows). At the tissue level, each ependymal cell coordinates the direction of their beating with that of neighboring cells (Figure 1C, grey arrows). The second feature, translational polarity, is unique to ependymal cells and refers to the clustering of cilia in a tuft. At the cellular level, this tuft is displaced relative to the center of the ependymal cell (Figure 1B, red arrow). At the tissue level, the positioning of the ciliary tuft is coordinated between adjacent cells (Figure 1C). Alteration of any of these polarities at either level impairs CSF flow circulation (Mirzadeh et al., 2010; Boutin et al., 2014; Guirao et al., 2010; Hirota et al., 2010; Ohata et al., 2014). Cilia axonemes arise from basal bodies (BB) which are cylindrical structures anchored perpendicular to the sub-apical surface of the cells (Figure 1D). BBs are polarized by the presence of appendices such as basal foot or striated rootlets. The basal foot protrudes in a direction correlated with the direction of cilia beating, while the striated rootlet protrudes in the opposite direction of cilia beating (Marshall, 2008). The ‘en face view’ observation of BBs’ organization allows the visualization of ependymal polarities (Mirzadeh et al., 2010; Boutin et al., 2014). Here, we describe an immunofluorescence (IF) protocol for observation of ciliated cells in mouse brain ventricular lateral wall whole mounts (LWWM). This protocol can be used for classical confocal microscopy analysis. In addition, it is well suited for super-resolution STimulated Emission Depletion (STED) microscopy if observation of structures that have features which are smaller than the optical diffraction limit is needed. Finally, we describe a combination of antibodies that allow the concomitant observation, in a single sample, of ependymal polarities at the level of individual cilia, individual cells and at the tissue level.

In situ hybridization and immunostaining are common techniques for localizing gene expression, the mRNA and protein respectively, within tissues. Both techniques can be applied to tissue sections to achieve similar goals, but in some cases, it is necessary to use them together. For example, complement C1q is a secreted protein complex that can target the innate immune response during inflammation. Complement has been found to be elevated early and before severe neurodegeneration in several disease models. Thus, complement may serve as an important marker for disease progression and may contribute to the pathology under certain conditions. Since complement is a secreted complex, immunostaining for C1q does not necessarily reveal where compliment is produced. In situ hybridization for complement components, C1q a, b, or c mRNA, is ideal to mark complement producing cells in tissue. In situ hybridization can be coupled with cell-type-specific immunostaining for accurate identification of the cell types involved. Protein localization and mRNA localization together can reveal details as to the relationship between complement producing and complement target cells within disease tissues. Here we outline the steps for combined in situ hybridization and immunostaining on the same tissue section. The protocol outlined here has been designed for detection of complement C1q in neurons and microglia in the mouse brain.

Provided here are two approaches for combined ISH/IH. In the 1st example, in situ hybridization of C1q mRNA is performed together with fluorescent detection of Purkinje neuron cell bodies using Calbindin-D28K antibody. In the 2nd example, C1q mRNA in situ is performed together with 3,3’-diaminobenzidine (DAB) detection of microglia using CD68 antibody. Please note that modifications to the protocol may be needed for the use of distinct probes and antibodies, as well as alternate tissue-processing methods that are not specified herein. For appropriate examples of procedure results, please see images published in Lopez et al.. (2012).

This protocol comprises the entire process of immunofluorescence staining on mouse cochlea whole mount, starting from tissue preparation to the mounting of the tissue. This technique provides “three-dimensional” views of the stained components in order to determine the localization of a protein of interest in the tissue in its natural state and environment.

This protocol comprises the entire process of immunofluorescence staining on mouse embryonic brains, starting from tissue preparation to mounting of the tissue sections.