At designated time point, mice were perfused with 4% polyformaldehyde (PFA) (20–30 mL) before dissection of brain tissue. The brain tissue was dehydrated with 30% sucrose 24 h before being embedded in Tissue Tek OCT (Sakura Finetek), followed by snap freezing in liquid nitrogen. The OCT embedded tissue blocks were kept at −80 °C for cryosection. The obtained brain slices (15 µm-thick) were fixed in Z-fix solution (Anatech) for 10 min, and permeabilized with 0.1% Triton X-100 for 5 min. To stain the mature neuron cells at the periphery of TBI site, a mature neuron marker, NeuN, was chosen. Anti-NeuN primary antibody was obtained from Cell Signaling Technology (1:150, Cat# 12943). The Cy3-conjugated donkey anti-rabbit secondary antibody (1:200, Cat# 711165-152, Jackson ImmunoResearch) was used. The nuclei were counterstained with DAPI using Vectashield mounting medium (Vector Lab). The images were taken from Olympus X81 fluorescence microscope.
Anti neun primary antibody
Manufactured by Cell Signaling Technology
The Anti-NeuN primary antibody is a tool used in research applications to detect the presence of the NeuN protein, a widely used marker for neuronal cell nuclei. This antibody specifically binds to the NeuN protein, allowing for the identification and visualization of neuronal cells in various experimental settings.
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Lab products found in correlation
Cy3 conjugated donkey anti rabbit secondary antibody, by Jackson ImmunoResearch (1 mentions)
X81 fluorescence microscope, by Olympus (1 mentions)
Vectashield mounting medium, by Vector Laboratories (1 mentions)
Tissue tek oct, by Sakura Finetek (1 mentions)
Z fix solution, by Anatech (1 mentions)
Confocal microscope, by Zeiss (1 mentions)
Alexa fluor 488 donkey anti rabbit secondary antibody, by Thermo Fisher Scientific (1 mentions)
Donkey anti goat alexa fluor 594 secondary antibody, by Thermo Fisher Scientific (1 mentions)
Freezing microtome, by Leica (1 mentions)
Uorescence microscopy, by Olympus (1 mentions)
Tunel kit, by Roche (1 mentions)
3 protocols using anti neun primary antibody
1
Mature Neuron Labeling in TBI Mouse Model
2
Visualizing Neuronal Calcium-Binding Protein
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After fixation, the brain and the spinal cord were immersed in a 30% sucrose solution until it sank to the bottom. Serial sections (50 μm thick) of the brain and the spinal cord were prepared using a freezing microtome (Leica, Germany). The sections were collected in phosphate buffer saline (PBS) for immunofluorescence staining. The sections were incubated with anti-CB primary antibody (1:600 dilution, List Biological Labs) at 4°C for overnight, followed by incubation with donkey anti-goat Alexa Fluor 594 secondary antibody (1:200 dilution, Life Technologies). Some of the sections were retained for CB-HRP/NeuN double immunofluorescence staining. The CB-HRP stained sections were incubated with anti-NeuN primary antibody (1:50 dilution, Cell Signaling Technology) at 4°C overnight, followed by incubation with donkey anti-rabbit Alexa Fluor 488 secondary antibody (1:200 dilution, Life Technologies). The sections were then mounted in sequence on the slides and counterstained with DAPI before covering with coverslips. The sections were observed and images were captured on a confocal microscope (Zeiss, Germany) using uniform parameters. After capturing the images, some sections were reused for Nissl staining to observe the neural morphology and Nissl body distributions.
3
Neuronal Apoptosis Detection Protocol
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After anesthesia, the rats were perfused transcardially with 4℃ PBS followed by 4% paraformaldehyde. Then, the brain tissue was taken out immediately and xed in 4% paraformaldehyde overnight. Sucrose solution was dehydrated in gradient, after rinsed, the brain tissue was quickly frozen on the machine.
Finally, the continuous coronal frozen section was made. TUNEL and staining for NeuN, a neuronal marker, were used together to detect neuronal apoptosis. Brie y, frozen sections were rewarmed at room temperature for 20 min, blocked with 5% sheep serum(Equitech-Bio,SS-0100) for 1 h and then incubated with anti-NeuN primary antibody (Cell Signaling Technology, cat# 12943, 1:200 dilution). A TUNEL kit (Roche, cat# 11684795910) was used to label apoptotic cells after the use of anti-NeuN secondary antibody (Alexa Fluor® Plus 594-conjugated). The sections were incubated with 50 μL of TUNEL reaction mixture (enzyme solution:labeling solution = 1:9), incubated at 37°C in the dark for 60 min, and washed 3 times with PBS for 5 min each, then the sections were sealed and observed by uorescence microscopy (Olympus, Tokyo, Japan). TUNEL-positive cells in ve different elds were counted. The results are expressed as cells/mm2, and the apoptotic ratio was calculated as the number of apoptotic cells/the total cell number × 100%.
Finally, the continuous coronal frozen section was made. TUNEL and staining for NeuN, a neuronal marker, were used together to detect neuronal apoptosis. Brie y, frozen sections were rewarmed at room temperature for 20 min, blocked with 5% sheep serum(Equitech-Bio,SS-0100) for 1 h and then incubated with anti-NeuN primary antibody (Cell Signaling Technology, cat# 12943, 1:200 dilution). A TUNEL kit (Roche, cat# 11684795910) was used to label apoptotic cells after the use of anti-NeuN secondary antibody (Alexa Fluor® Plus 594-conjugated). The sections were incubated with 50 μL of TUNEL reaction mixture (enzyme solution:labeling solution = 1:9), incubated at 37°C in the dark for 60 min, and washed 3 times with PBS for 5 min each, then the sections were sealed and observed by uorescence microscopy (Olympus, Tokyo, Japan). TUNEL-positive cells in ve different elds were counted. The results are expressed as cells/mm2, and the apoptotic ratio was calculated as the number of apoptotic cells/the total cell number × 100%.
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