Mice (24 h after reperfusion) and rats (21 days after reperfusion) were anesthetized with chloral hydrate and perfused transcardially with ice-cold saline followed by perfusion with 4% paraformaldehyde. The brains were removed and dehydration with a 10%, 20%, 30% sucrose gradient and then coronal sectioned (30 μm) using a vibrating microtome (Leica, Wetzlar, Germany). The sections were incubated in PBS containing 0.5% Triton X-100 and 10% normal goat serum for 1 h at room temperature, following by incubation with rabbit polyclonal NeuN (1:500; Abcam, Cambridge, UK) and rabbit polyclonal Iba1 (1:500; Wako, Osaka, Japan) at 4 °C overnight. After several PBS rinses, sections were incubated with Alexa Fluor 594 donkey anti-rabbit IgG (1:500; Invitrogen, Carlsbad, CA, USA) or Alexa Fluor 488 donkey anti-rabbit IgG (1:500; Invitrogen, Carlsbad, CA, USA). The number of NeuN or Iba1 positive cells was analysed by fluorescence confocal microscopy (EX61, Olympus, Tokyo, Japan). The positive cells were counted in three randomly chosen squares of identical size (460 × 460 μm) located in the cortical penumbra or hippocampal CA1 area.
Neuronal nuclei (neun)
Manufactured by Olympus
Sourced in Japan
NeuN is a monoclonal antibody that binds to the neuron-specific nuclear protein NeuN, which is expressed in most neuronal cell types. It is commonly used as a marker for identifying and quantifying neurons in various tissue samples and experimental models.
Automatically generated - may contain errors
Lab products found in correlation
Neuronal nuclei (neun), by Merck Group (2 mentions)
Ax 51 epifluorescence microscope, by Olympus (2 mentions)
Goat polyclonal antiserum against pgc 1α and nrf1, by Santa Cruz Biotechnology (2 mentions)
Alexa fluor 488 donkey anti rabbit igg, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 594 donkey anti rabbit igg, by Thermo Fisher Scientific (1 mentions)
Vibrating microtome, by Leica (1 mentions)
Goat anti rabbit igg conjugated with alexa fluor 488, by Thermo Fisher Scientific (1 mentions)
Goat anti mouse igg conjugated with alexa fluor 568, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 568, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (1 mentions)
Goat anti rabbit igg, by Thermo Fisher Scientific (1 mentions)
Goat anti mouse igg, by Thermo Fisher Scientific (1 mentions)
Cd206, by Olympus (1 mentions)
Cd16 32, by Abcam (1 mentions)
Cd206, by Abcam (1 mentions)
4 protocols using neuronal nuclei (neun)
1
Quantifying Neuronal and Microglial Responses
2
Dual Immunofluorescence Detection of PGC-1α and NeuN
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Double immunofluorescence staining [5 (link),7 (link),8 (link),43 (link),56 (link)] was carried out using a goat polyclonal antiserum against PGC-1α and NRF1 (Santa Cruz Biotechnology) and a mouse monoclonal antiserum against a specific neuronal marker, neuron-specific nuclear protein (NeuN; Chemicon). The secondary antisera included goat anti-rabbit IgG conjugated with AlexaFluor 488 and goat anti-mouse IgG conjugated with Alexa Fluor 568 (Molecular Probes, Eugene, OR, USA). Sections were viewed under an Olympus AX-51 epifluorescence microscope (Olympus, Kyoto, Japan); immunoreactivity for NeuN exhibited red fluorescence and PGC-1α manifested green fluorescence.
3
Double Immunofluorescence Staining for PGC-1α and NRF1
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According to our previous studies [15 (link),74 (link)], double immunofluorescence staining was accomplished by using a goat polyclonal antiserum against PGC-1α and NRF1 (Santa Cruz Biotechnology), and a mouse monoclonal antiserum against neuron-specific nuclear protein (a specific neuronal marker, NeuN; Chemicon). The secondary antisera contained a goat anti-rabbit IgG conjugated with AlexaFluor 488 and a goat anti-mouse IgG conjugated with Alexa Fluor 568 (Molecular Probes, Eugene, OR, USA). Tissue sections were examined under an Olympus AX-51 epifluorescence microscope (Olympus, Kyoto, Japan); immunoreactivity for NeuN exhibited red fluorescence, and PGC-1α and NRF1 manifested green fluorescence.
4
Quantitative Immunohistochemistry Analysis of Microglia and Neurons
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Immunohistochemistry analysis was performed on 30-μm free-floating sections [6 (link)]. The sections were incubated in PBS containing 0.5% Triton X-100 and 10% normal goat serum for 1 h at room temperature and then with mice monoclonal Iba1 (1:500; Abcam, Cambridge, UK), rabbit polyclonal CD16/32 (1:500; Abcam, Cambridge, UK), rabbit polyclonal CD206 (1:500; Abcam, Cambridge, UK) and mice monoclonal NeuN (1:500; Abcam, Cambridge, UK) at 4 °C overnight. After several PBS rinses, sections were incubated with Alexa Fluor 488 donkey anti-mice IgG (1:500; Invitrogen, Carlsbad, CA, USA) or Alexa Fluor 596 donkey anti-rabbit IgG (1:500; Invitrogen, Carlsbad, CA, USA). An experimenter (Z.H.J) coded all slides from the experiments before quantitative analysis. The number of Iba1/CD16/32-, Iba1/CD206- or NeuN-positive cells was analyzed using fluorescence confocal microscopy (EX61, Olympus, Tokyo, Japan) by another experimenter (X.L.X) blinded to the study code. The means were calculated from 3 randomly selected microscopic areas in the cortex and striatum of each section, and 5 consecutive sections were analyzed for each brain. The data are expressed as the mean numbers of cells per square millimeter.
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