Immunofluorescence protocol for localizing protein targets in brain tissue from diverse model and non-model mammals

Abstract

Previous immunostaining protocols are highly specific for model organisms and often not suitable for diverse specimens that are non-perfused and over-fixed (i.e., tissues sitting in fixatives for months/year). Here, we present an immunofluorescence protocol for localizing protein targets in brain tissue from 11 model and non-model mammals. We describe preparation of both fresh and fixed tissues including steps for deparaffinization, fixation, and cryoprotection. We then detail immunofluorescence procedures including antigen retrieval, reducing autofluorescence, nuclear staining, mounting, and image collection.

Keywords: Evolutionary Biology; Neuroscience.

Graphical abstract

Figure 1

Examples of outcome for single immunostainings performed on a wide range of species The same immunostaining was first tested on a wide variety of species. (A) On the left, evolutionary tree of the species tested with this protocol. This tree was created with https://phylot.biobyte.de, species taxonomy codes were obtained from NCBI taxonomy database. Scientific names correspond to the following common names: Mus musculus (mouse), Macaca mulatta (Rhesus macaque), Macaca fascicularis (crab-eating macaque), Homo sapiens (human), Pan troglodytes (chimpanzee), Carollia perspicillata (Seba’s short-tailed bat), Bos taurus (cattle), Tursiops truncatus (common bottlenose dolphin), Panthera leo (lion), Panthera tigris (tiger), Panthera pardus (leopard). On the right, indication of the correspondent orders. Color coded is as follows: Rodentia in gray; Primates in dark yellow; Chiroptera in purple; Artiodactyla in blue; Carnivora in orange. (B–J) In green, immunostaining for Glial fibrillary acidic protein (GFAP) labeling astrocyte intermediate filament-III; in blue, NeuroTrace labeling nuclei. Species of the brain sections are indicated per each micrograph. Each micrograph is color labeled depending on the order the species belongs to. Scale bars = 20 μm.

Figure 2

Examples of outcome for double immunostaining tested on a wide range of species Different examples of double immunostaining on prefrontal cortex specimens from a selected species (i.e., cattle: Bos taurus). The same double immunostaining has been performed on the same species as in Figure 1A, not showed. (A–A″) NeuroTrace (NT) in blue; S100b (a calcium binding protein expressed in an astrocyte subpopulation) in green; GFAP in red. (B–B″) NT in blue, Aldh1l1 (a pan-astrocytic marker) in green; GFAP in red. Scale bars = 20 μm. (C–C″) NT in blue, CD31 (a marker for endothelial cells) in green; GFAP in red. Full arrowheads point to double-stained cells; empty arrowheads point to single-stained cells. Scale bars = 20 μm.