Optimized Immunostaining of Embryonic and Early Postnatal Mouse Brain Sections.

Kawssar Harb,Michele Bertacchi,Michèle Studer

doi:10.21769/bioprotoc.3868

Abstract

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).

Highlights

An example of counting analysis showing the percentage of marker-positive cells normalized to the total number of DAPI or Hoechst-positive cells
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
We present a protocol that worked in our hands for most of the antibodies aimed at detecting key determinants of neocortical development in thick vibratome or thin cryostat sections

Summary

Procedure

6. Wash the brains 3 times for 10 min each in PBS at 4 °C (on shaker). 3. The following day add 500 μl of primary antibodies by diluting them to the appropriate concentration with the antibody solution (3% GS, 3% BSA, 0.3% Triton). 9. Lay your slides horizontally and avoid the sections to dry by immediately adding the blocking solution (10% GS, 0.3% Triton in PBS). Prepare 200-300 μl of primary antibodies for each slide by diluting them to the appropriate concentration with the antibody solution (3% GS, 0.3% Triton in PBS). Prepare 200-300 μl of secondary antibodies per slide by diluting them to 1/300 in the antibody solution (3% GS, 0.3% Triton). Add 200-300 μl of the diluted secondary antibodies to the slides and cover them with cover slips to homogeneously distribute the antibody.

Data analysis for thick vibratome sections

Troubleshooting 2

Findings

Tips 2