Immunohistochemistry/IF - mouse brain Annotation Reliability score

Brain methods

Immunohistochemistry/IF - mouse brain

As a complement to the immunohistochemically stained tissues, the protein atlas also includes the mouse brain atlas as a sub compartment of the normal tissue atlas. In which comprehensive profiles are available in mouse brain. A selected set of targets have been analyzed by using the antibodies in serial sections of mouse brain which covers 129 areas and subfields of the brain, several of these regions difficult to cover in the human brain. In addition pituitary, retina and trigeminal ganglions are included in recent and future image series but not annotated yet.

The tissue microarray method used within the human protein atlas enabled the global mapping of proteins in the human body, including the brain. Currently, the human tissue atlas covers four areas of the human brain: cerebral cortex, hippocampus, caudate and cerebellum. Due to the heterogeneous structure of the brain, with many nuclei and cell-types organized in complex networks, it is difficult to achieve a comprehensive overview in a 1 mm tissue sample. Analysis of more human brain samples, including smaller brain nuclei, is thus desirable in order to generate a more detailed map of protein distribution in the brain. Therefore, we here complemented the human brain atlas effort with a more comprehensive analysis of the mouse brain. A series of mouse brain sections is explored for protein expression and distribution in a large number of brain regions.

Antibodies are selected against protein involved in normal brain physiology, brain development and neuropathological processes. A limit of 60% homology (human vs mouse) is used as cut off when comparing the PrEST sequence for the antibody targets.

Selected antibodies are applied to test-sections containing brain regions or cell types with known expression based on in situ hybridization (Allen Brain Atlas) and single cell RNAseq data (Linnarsson Lab and Barres Lab). Staining patterns are evaluated based on consistency between staining patterns of multiple antibodies against the same target and match to transcriptomics data. Antibody immunoreactivity is visualized using tyramid signal amplification shown in green. A nuclear reference staining (DAPI) is visualized in blue. The immunofluorescence protocol is standardized through antibody concentration and incubation time are variable depending on protein abundance and antibody affinity determined during the test staining. The complete mouse brain profile is represented by serial coronal sections of adult mouse brain, 16 µm thick. Stained slides are then scanned and digitalized before further processing.

Table 1. Brain regions. Abbreviations are based on The Mouse Brain in Stereotaxic Coordinates, Third Edition: The coronal plates and diagrams (ISBN: 9780123742445)

Annotation

The digitalized images are processed (axel-adjusted and tissue edges defined) and regions of interest (ROIs) are then marked according to the table above. These ROIs are then used for image analysis and the relative fluorescence intensity is listed for each region. The relative fluorescence is defined intensity of the annotated region relative to the intensity of the region with highest intensity.

The overview and preserved orientation in the mouse brain has enabled us to annotate additional cell classes (ependymal), glial subpopulations (microglia, oligodendrocytes, and astrocytes), and additional brain specific subcellular locations (axon, dendrite, synapse, and glia endfeet) for each investigated protein.

All images of immunofluorescence stained sections were manually annotated by specially educated personnel followed by review and verification by a second qualified member of the staff. The cellular and subcellular location of the immunoreactivity is defined and a summarizing text is provided describing the general staining pattern.

Specificity is validated by comparing the data with in situ hybridization data (Allen brain atlas) and/or available literature; support from other data leads to a supportive reliability score, while more unknown targets are viewed as uncertain and awaits further validation.

Reliability score

A reliability score is set for all genes and indicates the level of reliability of the analyzed protein expression pattern based on available protein/RNA/gene characterization data.

The reliability score of the antibodies in mouse brain atlas is scored as Supported or Uncertain depending on support from in situ hybridization data (Allen brain atlas) and/or previous published data, UniProtKB/Swiss-Prot database.