Tissue was dissociated with FACS lysis buffer (final concentration: 0.32M Sucrose, 10mM Tris − HCl pH8.0, 5mM CaCl2, 3mM Mg(acetate)2, 0.1mM EDTA, 1mM DTT, 0.3% Triton-X −100 and 100× PIC) into single cell suspension, then fixed with 1% formaldehyde for 5 mins, and stop by 0.125M Glycine. Then, cells were washed twice with cold 1xPBS to remove excessed formaldehyde and glycine. After incubating with blocking buffer (Final concentration: 10% normal goat serum, 5% BSA, 0.1% Triton-X-100 and 1XPIC) for half-hour, cells were double-labeled with Arc antibody (Bioss) in 1:20000 dilution per million cell and NeuN antibody (Abcam) in 1:20000 per million cell, together with DAPI (Thermofisher) in 1:2000. PBPT buffer was used for washing (twice each time) and resuspend into 500ul 1x PBS for FACS sorting. FACS was performed on a BD FACSArial (BD Science), and cells were sorted into lysis buffer from Arcturus PicoPure RNA isolation kit (Applied Biosystems). Then, RNA was isolated from sorted cells by Arcturus PicoPure RNA isolation kit (Applied Biosystems) following the manufacturers protocol. 5ng of total RNA per sample and SMRTer Stranded Total RNA-seq Kit v2 Pico Input Mammalian Components kit (Clontech) was used for RNA-seq library preparation following the manufacturers protocol. Sequencing was conducted at GENEWIZ (Suzhou, China) with 150pb Pair-End reads.
Neun antibody
Manufactured by Abcam
Sourced in China, United Kingdom
The NeuN antibody is a protein marker commonly used to identify neuronal nuclei in various tissues. It binds to the neuronal-specific nuclear protein NeuN, which is expressed in most neuronal cell types. This antibody is a valuable tool for researchers studying the nervous system and neurological disorders.
Automatically generated - may contain errors
Lab products found in correlation
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (5 mentions)
Arc antibody, by Bioss Antibodies (2 mentions)
Facs arial, by BD (2 mentions)
Smrter stranded total rna seq kit v2 pico input mammalian components kit, by Takara Bio (2 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Alexa fluor 488 donkey anti mouse igg, by Thermo Fisher Scientific (1 mentions)
U rfl t, by Olympus (1 mentions)
Live dead cell staining kit, by Abcam (1 mentions)
Triton x 100, by Merck Group (1 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (1 mentions)
Triton x 100, by Avantor (1 mentions)
Confocal microscope, by Leica Microsystems (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Bioss Antibodies (1 mentions)
Gfap antibody, by Merck Group (1 mentions)
Cxcr2 antibody, by Abcam (1 mentions)
Cd34 antibody, by Boster Bio (1 mentions)
Alexa fluor 546 conjugated donkey anti goat, by Jackson ImmunoResearch (1 mentions)
α btx, by Thermo Fisher Scientific (1 mentions)
Reep1 antibody, by Merck Group (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions)
19 protocols using neun antibody
1
Single-cell RNA-seq of FACS-sorted Arc+ Neurons
2
Live-Dead Cell Staining and NeuN Immunodetection
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Live-Dead cell staining kit was used according to the manufacturer’s instructions (Abcam). Briefly, after 0, 7, and 14 days of monoculture or 7, 14, and 21 days of coculture, samples were labeled with mixture of solution A (Live-Dye) and solution B (PI) in the staining buffer for 15 min at 37 °C. The samples were then washed once with the staining buffer and examined under Olympus IX71 with U-RFL-T (detects FITC and TRITC). For NeuN immunostaining, cells were fixed with 4% paraformaldehyde (PFA) and treated with 0.2% Triton X-100 (Sigma), followed by blocking in 4% BSA (Millipore) at 4 °C overnight. Cells were then labeled with NeuN antibody (1:500, Abcam) for 24 hr at 4 °C, and were stained with Alexa Fluor® 488 donkey anti-mouse IgG (1:500, Invitrogen) for 1 hr at room temperature, followed by DAPI (Life Technologies) staining. The number of stained cells was counted from five random fields (Live-Dead cell, NeuN staining) of view under the microscope and statistical analysis on the cell viability was performed.
3
Visualization of TRPV3 Expression in Cells
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HEK 293T cells were transfected with mouse-TRPV3-EGFP plasmid using Lipofectamine 2000 (Invitrogen) following the manufacturer's instructions and were maintained at 37 °C under 5% CO2 in DMEM supplemented with 10% fetal bovine serum. HEK 293T cells expressing TRPV3 and mouse cortical neurons in a 15 mm confocal culture dish were fixed in 4% paraformaldehyde for 15 min at 4 °C after removal of culture medium. The cells were added with 0.3% Triton X-100 (Amresco) in PBS for 15 min and blocked by 10% sheep serum in 0.01 mol/L PBS for 1 h at room temperature before incubated with primary antibodies overnight at 4 °C. The rabbit monoclonal NeuN antibody (1:1000; Abcam), mouse monoclonal TRPV3 antibody (1:1000; Abcam). The immunoreactivity was visualized with Alexa Fluor 488- or 594-conjugated secondary antibodies (1:200; ZSGB-BIO). Immunocytochemistry was used for visualization of TRPV3 proteins in cells using a confocal microscope (Leica Microsystems).
4
Immunofluorescence Staining of Hydrogel Tissues
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Immunofluorescence staining and double/ triple immunostaining were performed as previously described [12 (link)]. Briefly, the polyacrylamide hydrogel (PAG) tissues were harvested and cryosectioned at 35 μm. The sections were first incubated with 5% normal donkey serum in 0.1 M PBS for 2 h at room temperature. The sections were then incubated overnight at 4 °C with the following primary antibodies: CXCL1 antibody (1:100, rabbit; Boster), phosphor-NF-кB p65 (Ser536) (pNF-кB) antibody (Rabbit, 1:500; Sigma), CXCR2 antibody (1:100, rabbit; Abcam), GFAP antibody (1:1000, mouse; Sigma), CD11 antibody (1:250, mouse; Abcam), and NeuN antibody (1:300, mouse; Abcam). The sections were then incubated for 1 h with FITC- or Cy3-conjugated secondary antibodies (1500, Abcam) at room temperature. For double immunofluorescence, sections were incubated with a mixture of primary antibodies at 4 °C overnight, followed by a mixture of FITC- and Cy3-conjugated secondary antibodies. Sections were coverslipped using a water-based mounting medium containing DAPI (Bioss, China). The stained sections were examined using an Olympus fluorescence microscope, analyzed by Image Pro-plus 6.0 (Image Pro-Plus Kodak, USA), and images were taken with a CCD Spot camera. Negative controls in which the primary antibody was omitted or replaced with PBS were used to confirm immunospecificity.
5
Immunofluorescence Staining of Cerebellum
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Sections were processed for immunofluorescence staining with CD34 antibody to label microvessels, NeuN antibody to label neurons, Iba1 and GFAP antibodies to label microglia and astrocytes. Briefly, sections were boiled to recovered antigen in 0.01 mol/L citrate buffer (pH 6.0) at 96°C to 98°C for 15 minutes. Nonspecific binding sections were blocked with 5% normal goat serum or donkey serum in PBS at room temperature for 2 hours. Sections were then incubated with CD34 antibody (from rabbit, 1 : 200, Boster, Wu Han, China) and NeuN antibody (from rabbit, 1 : 1,000, Abcam, British) separately; Iba1 antibody and GFAP antibody (from goat or from mouse, 1 : 1,000, Abcam, Cambridge, MA, USA) for double-labelling overnight at 4°C. After PBS washes, the sections were incubated with secondary antibody (Alexa Fluor 488-conjugated goat anti-rabbit lgG or donkey anti-rabbit, or donkey anti-mouse and Alexa Fluor 546-conjugated donkey anti-goat, Jackson Immunoresearch, West Grove, PA, USA) at 37°C for 2 hours. The concentration of all secondary antibodies was 1 : 1,000, then washed in PBS three times (5 minutes each) and covered with anti-quenching fluorescence mounting medium. The sections of cerebellum were performed a regular HE staining process.
6
Single-cell RNA-seq of FACS-sorted Arc+ Neurons
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Tissue was dissociated with FACS lysis buffer (final concentration: 0.32M Sucrose, 10mM Tris − HCl pH8.0, 5mM CaCl2, 3mM Mg(acetate)2, 0.1mM EDTA, 1mM DTT, 0.3% Triton-X −100 and 100× PIC) into single cell suspension, then fixed with 1% formaldehyde for 5 mins, and stop by 0.125M Glycine. Then, cells were washed twice with cold 1xPBS to remove excessed formaldehyde and glycine. After incubating with blocking buffer (Final concentration: 10% normal goat serum, 5% BSA, 0.1% Triton-X-100 and 1XPIC) for half-hour, cells were double-labeled with Arc antibody (Bioss) in 1:20000 dilution per million cell and NeuN antibody (Abcam) in 1:20000 per million cell, together with DAPI (Thermofisher) in 1:2000. PBPT buffer was used for washing (twice each time) and resuspend into 500ul 1x PBS for FACS sorting. FACS was performed on a BD FACSArial (BD Science), and cells were sorted into lysis buffer from Arcturus PicoPure RNA isolation kit (Applied Biosystems). Then, RNA was isolated from sorted cells by Arcturus PicoPure RNA isolation kit (Applied Biosystems) following the manufacturers protocol. 5ng of total RNA per sample and SMRTer Stranded Total RNA-seq Kit v2 Pico Input Mammalian Components kit (Clontech) was used for RNA-seq library preparation following the manufacturers protocol. Sequencing was conducted at GENEWIZ (Suzhou, China) with 150pb Pair-End reads.
7
Multiprotein Immunofluorescence in Neurons
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The α-BTX (tetramethylrhodamine conjugate) antibody (Invitrogen, T1175), synaptophysin antibody (Santa Cruz Biotechnology, sc-17750), Bip antibody (CST, 3177), choline acetyltransferase (Abcam, ab18736), REEP1 antibody (Sigma-Aldrich, SAB2101976), and NeuN antibody (Abcam, ab177487) were used for immunofluorescence. The REEP1 antibody (Sigma-Aldrich, SAB2101976) was also used for western blotting.
8
Detecting miR-21 Expression in Spinal Neurons
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To verify whether miR-21 is expressed in spinal cord neurons, ISH was performed on transverse frozen sections using the 30% LNA-70% DNA3′-DIG-labelled mercury probes (Exiqon, Vedbaedk, Denmark), according to the manufacturer’s protocol. Hybridized miR-21 was visualized with Alkaline Phosphatase (AP). After ISH, sections were blocked in PBS containing 0.5% BSA and 0.05% Tween 20 for immunostaining. Incubation with a NeuN antibody (1:500; Abcam) was followed by incubation with a fluorescent secondary antibody. Finally, cells were stained with DAPI (1:7500, Sigma, USA) for 10 min to visualize nuclei.
9
Immunofluorescent Staining of Paraformaldehyde-Fixed Cells
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Cells were fixed with 4% paraformaldehyde for 15 min at 37°C, washed three times with PBS, and then, incubated overnight at 4°C in PBS containing 2% Goat serum, 0.03% Triton X-100 in, p-MLKL antibody (CST, #37333, 1:1,200), and NeuN antibody (Abcam, ab177487, 1:500). They were then incubated for 2 h at 25°C with the following fluorescent secondary antibodies. Cell nuclei were stained with DAPI, and images were obtained using a fluorescence microscope (Olympus, Osaka, Japan).
10
Apoptosis Detection in ICH Model
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At 24 h after ICH, TUNEL staining was performed with an in situ apoptosis detection kit (Roche, Basel, Switzerland) according to the manufacturer’s instruction. For NeuN and TUNEL co-staining, the sections were first labeled with a NeuN antibody (1:400, Abcam), followed by TUNEL. The slides were analyzed using a fluorescence microscope (Bx51, Olympus).
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