Ab104224

Manufactured by Abcam

Sourced in United States, United Kingdom, China, Japan

Ab104224 is a recombinant protein produced in Escherichia coli. It is a fusion protein containing a glutathione S-transferase (GST) tag.

Automatically generated - may contain errors

Lab products found in correlation

Ab7260, by Abcam (13 mentions) Ab5076, by Abcam (13 mentions) Ab53554, by Abcam (9 mentions) Ab1211, by Abcam (7 mentions) Ab150113, by Abcam (6 mentions) Ab177487, by Abcam (6 mentions) Ab10062, by Abcam (6 mentions) Ab4648, by Abcam (5 mentions) Fluorescence microscope, by Leica (4 mentions) Ab108319, by Abcam (4 mentions) Ab192890, by Abcam (4 mentions) Ab18258, by Abcam (4 mentions) Ab150077, by Abcam (4 mentions) In situ cell death detection kit, by Roche (4 mentions) Ab183830, by Abcam (4 mentions) 4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (4 mentions) Ab32127, by Abcam (3 mentions) Ab178846, by Abcam (3 mentions) Sc 56036, by Santa Cruz Biotechnology (3 mentions) 4 6 diamidino 2 phenylindole (dapi), by Vector Laboratories (3 mentions)

151 protocols using ab104224

Immunofluorescence Staining of Retinal Cells

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Frozen retinal sections were prepared and incubated with mouse antibody against NeuN (Abcam, Cambridge, UK, #ab104224; 1:200) at 4 °C overnight. The sections were then stained with the secondary antibody anti-mouse Alexa Fluor 594 (Abcam, Cambridge, UK, #ab150116; 1:200) for 2 h at room temperature, followed by 4′,6-diamidino-2-phenylindole (DAPI) staining of 5 min to label cell nuclei.
RMCs and RGCs seeded onto coverslips were fixed with 4% paraformaldehyde for 20 min. The following primary antibodies were used: mouse anti-GS (#ab64613; 1:200), rabbit anti-IL-17RA (#ab180904; 1:200), mouse anti-NeuN (#ab104224; 1:200) (all from Abcam, Cambridge, UK), and chicken anti-NeuN (Novus Biologicals, Littleton, CO, USA, #NBP2-10491; 1:200). Alexa Fluor 488 (#ab150113), 594 (#ab150080), or 405 (#ab175674) conjugated secondary antibodies (all from Abcam, Cambridge, UK; 1:200) were followed to incubate these cells. In RMCs, GS⁺ cells and GS⁺IL-17RA⁺ cells in five visual fields of a coverslip were calculated into an average number, respectively. The average number of GS⁺IL-17RA⁺ cells in a coverslip was reported as a percentage of the average number of GS⁺ cells in statistical analysis.

Qiu A.W., Huang D.R., Li B., Fang Y., Zhang W.W, & Liu Q.H. (2021). IL-17A injury to retinal ganglion cells is mediated by retinal Müller cells in diabetic retinopathy. Cell Death & Disease, 12(11), 1057.

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Anti-Inflammatory and Neuroprotective Assay

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LPS (#L3129) were purchased from Sigma-Aldrich (WI, USA), and 4-OI (#HY-112675) was purchased from MCE (NJ, USA) and dissolved in DMSO (Solarbio, #D8371, Beijing, China). The primary antibody against iNOS (18985-1-AP), Bax (50599-2-Ig), Nrf2 (16396-1-AP), Lamin B (66095-1-Ig), GAPDH (60004-1-Ig), IL-6 (66146-1-Ig), NQO1 (67240-1-Ig), Arg-1 (66129-1-Ig) and CD68 (28058-1-AP) were obtained from ProteinTech (Wuhan, China). The primary antibodies against HO-1 (#43966), COX2 (#12282), Irg-1 (#17805), Iba-1 (#17198), and Cleaved Caspase3 (#9661) were purchased from CST (MA, USA), and the primary antibodies against NeuN (ab104224), Bcl2 (ab196495) and Alexa Fluor 488-labeled and Alexa Fluor 594-labeled goat anti-rabbit/mouse secondary antibodies were purchased from Abcam (Cambridge, UK). Finally, 4′,6-diamidino-2-phenylindole (DAPI) was purchased from Yeasen Biochemical (Shanghai, China).

Ni L., Xiao J., Zhang D., Shao Z., Huang C., Wang S., Wu Y., Tian N., Sun L., Wu A., Zhou Y., Wang X, & Zhang X. (2022). Immune-responsive gene 1/itaconate activates nuclear factor erythroid 2-related factor 2 in microglia to protect against spinal cord injury in mice. Cell Death & Disease, 13(2), 140.

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Multimodal Immunohistochemical Profiling of Cellular Markers

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For immunohistochemistry, mice were perfused intracardially with 4% paraformaldehyde phosphate buffer. Serial coronal sections were prepared and blocked by PBS containing 3% BSA and 0.3% Triton‐X100 and then incubated with primary antibodies overnight at room temperature as the following: rabbit anti‐Axin2 (Abcam, ab109307, 1:200), rat anti‐MBP (Merck Millipore, MAB386, 1:500), mouse anti‐MT1 receptor (Santa cruz, sc‐390328, 1:50), mouse anti‐NeuN (Abcam, ab104224, 1:600), rabbit anti‐NG2, (Abcam, ab255811, 1:150), rabbit anti‐GFAP (Abcam, ab207165, 1:500), mouse anti‐Neurofliment (Abcam, ab7795, 1:200), rabbit anti‐Olig2(Abcam, ab109186, 1:200), mouse anti‐CC1(Sigma‐Aldrich, OP80, 1:100), mouse anti‐Flag (Sigma‐Aldrich, F1804, 1:100), Rabbit anti‐β‐catenin (Abcam, ab16051, 1:200), Rabbit anti‐vglut1 (Synaptic Systems Cat. No.135308, 1:200). After washing with PBS, corresponding secondary antibodies conjugated with Alexa Fluro 488 (donkey anti‐rabbit, donkey anti‐mouse, Jackson ImmunoResearch, 1:500, AB_2313584, AB_2338845) or Alexa Fluro 594 (donkey anti‐rat, donkey anti‐mouse, donkey anti‐rabbit, Jackson ImmunoResearch, Jackson 1:500, RRID:AB_2338372, AB_2338871, AB_2338059) were incubated with the sections for 2–4 h at room temperature protected from light. After washing with PBS, sections were counterstained with Hoechst33324 (1:1000, Sigma) for 20 min.

Liang L., Zeng T., Zhao Y., Lu R., Guo B., Xie R., Tang W., Zhang L., Mao Z., Yang X., Wu S., Wang Y, & Zhang H. (2021). Melatonin pretreatment alleviates the long‐term synaptic toxicity and dysmyelination induced by neonatal Sevoflurane exposure via MT1 receptor‐mediated Wnt signaling modulation. Journal of Pineal Research, 71(4), e12771.

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Immunocytochemistry of Hippocampal Neurons

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Primary hippocampal neuronal cultures grown on glass coverslips for 7 days were fixed using 2% paraformaldehyde and permeabilized using phosphate-buffered saline (PBS) containing 0.5% Triton X-100 (Sigma-Aldrich). The cells were then blocked using 2% bovine serum albumin in PBS containing 0.1% Triton X-100 (PBS-T) before applying the primary antibodies against NeuN (1 : 200, cat#ab104224, RRID:AB_10711040) and ALK2 (1 : 200, cat#ab60157, RRID:AB_940117, both Abcam). After overnight incubation, the cultures were washed with PBS-T and incubated for 1 h with secondary antibodies, followed by nuclear staining with Hoechst 33342 dye (Invitrogen). Coverslips were mounted using Immu-mount medium (ThermoFisher, Mississauga, Ontario, Canada) and the images were collected using an LSM 510 confocal microscope (Zeiss, Toronto, Ontario, Canada), equipped with a 10×/0.3 objective.

Beraldo F.H., Ostapchenko V.G., Xu J.Z., Di Guglielmo G.M., Fan J., Nicholls P.J., Caron M.G., Prado V.F, & Prado M.A. (2018). Mechanisms of neuroprotection against ischemic insult by stress–inducible phosphoprotein–1/prion protein complex. Journal of neurochemistry, 145(1), 68-79.

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Quantifying Neuronal Apoptosis in Cortex

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The TUNEL and NeuN co-staining was conducted to measure neuronal apoptosis/neuronal death as previous[26 (link)]. The 10-μm coronal frozen slices were first incubated with anti-NeuN antibody (ab104224, Abcam, Cambridge, MA, USA) at 4 °C overnight. On the second day, the brain slices were further stained using In Situ Apoptosis Detection Kit (12156792910 Roche, MO, USA) based on manufacture’s instruction. The images were visualized and photographed using a fluorescence microscope (Leica Microsystems, Wetzlar, Germany). Quantification of positive cells was counted in three randomized areas chosen in the ipsilateral cortex.

Fang Y., Shi H., Huang L., Ren R., Lenahan C., Xiao J., Liu Y., Liu R., Sanghavi R., Li C., Chen S., Tang J., Yu J., Zhang J.H, & Zhang J. (2021). Pituitary Adenylate Cyclase-Activating Polypeptide Attenuates Mitochondria-Mediated Oxidative Stress and Neuronal Apoptosis after Subarachnoid Hemorrhage in Rats. Free radical biology & medicine, 174, 236-248.

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Comprehensive Western Blot Analysis of Neural Markers

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Western blotting was performed as previously described (Huang et al. 2018 (link)) with the diluted primary antibodies NeuN (1: 300, cat. # ab104224, Abcam), GFAP (1: 300, cat. # ab7260, Abcam), Iba-1 (1: 300, cat. # ab178846, Abcam), caspase-1 (1: 100, cat. # sc-56036, Santa Cruz), NLRP3 (1: 1,000, cat. # DF7438, Affinity), ASC (1: 1,000, cat. # ab47092, Abcam), Gasdermin D (GSDMD, 1: 500, cat. # sc-393581, Santa Cruz), IL-1β (1: 1,000, cat. # ab254360, Abcam), IL-18 (1: 1000, cat. # ab207323, Abcam), BDNF (1: 1000, cat. # ab108319, Abcam), PSD95 (1: 1000, cat. # ab238135, Abcam), Syna (1: 1000, cat. # ab32127, Abcam), and GAP43 (1: 1000, cat. # ab75810, Abcam) and incubated for 12 h at 4 °C. After being washed three times with 0.1 M PBS, the membranes were incubated with HRP-conjugated anti-rabbit or anti-mouse secondary antibodies (diluted to 1: 5000, Abcam, USA) for 2 h at RT. Finally, the protein bands were visualized using a Western blotting detection kit (Wanleibio, China).

Dong D., Ren A., Yang Y., Su J., Liu L., Zhuo W, & Liang Y. (2022). VX-765 Alleviates β-Amyloid Deposition and Secondary Degeneration in the Ipsilateral Hippocampus and Ameliorates Cognitive Decline after Focal Cortical Infarction in Rats. Journal of Molecular Neuroscience, 72(12), 2389-2397.

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Immunohistochemical Analysis of Mouse Brain

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Mouse brains were perfused with 0.9% NaCl solution, fixed in 4% paraformaldehyde PBS solution, and dehydrated in 30% sucrose solution. After embedding, brain sectioning was done coronally. Primary antibodies mouse anti-Iba1 (1 : 500; Abcam Cat# ab15690, RRID:AB_2224403), and mouse anti-NeuN (1 : 100; Abcam Cat# ab104224, RRID:AB_10711040) were applied. Secondary antibodies, goat anti mouse Alexa Fluor 488 or 594 (1 : 500, EARTHOX, San Francisco, # E031220-01), were employed. Antiquenching mounting medium containing DAPI (VECTASHIELD, USA) was added to slides prior to being covered with coverslips for observation. Positive cells were detected by a fluorescence microscope (Olympus BX51, NIKON, Japan) at excitation/emission wavelengths of 547/570 nm (Cy3, red), 488/520 nm (FITC, green), and 360/460 nm (FITC, blue). Images were taken under 400x magnifications in 5-vision fields/section randomly, and the immunoreactive cells were counted.

Du X., Gao F., Chen S., Botchway B.O., Amin N., Hu Z, & Fang M. (2020). Combinational Pretreatment of Colony-Stimulating Factor 1 Receptor Inhibitor and Triptolide Upregulates BDNF-Akt and Autophagic Pathways to Improve Cerebral Ischemia. Mediators of Inflammation, 2020, 8796103.

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Immunofluorescence Staining of Spinal Cord

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Procedures were described in our previous study (Liu et al., 2022 (link)). Briefly, embedded spinal cord samples were cut into frozen sections with the thickness of 20 μm. Then samples were mounted onto gelatin-coated glass slides for immunofluorescence staining. The sections were blocked in normal donkey serum (5%) in Tris buffered saline tween (TBST) for 1 h at room temperature, and then incubated with corresponding primary antibodies overnight at 4°C. The primary antibodies were as follows: mouse anti-GFAP (1:1000, #c9205, Sigma), mouse anti-OX42 (1:1000, #ab1211, Abcam), mouse anti-NeuN (1:1000, #ab104224, Abcam), and rabbit anti-NLRP3 (1:200, #PA5-79740, Invitrogen). Then the sample sections were labeled with the fluorescent secondary antibodies (Cy3-, Cy5-, or FITC-conjugated) for 1 h at room temperature after washing in the dark and counterstained with DAPI. The pictures of sample sections were captured by laser scanning confocal microscope (Nikon A1R, Japan). Images were captured with uniformed settings and experimenters were blinded to treatment groups for image quantification. Three to five random images from per rat tissue in each group were selected, averaged, and then compared as described previously (Liu et al., 2016 (link); Li et al., 2021 (link)).

Zhang Y., Chen R., Hu Q., Wang J., Nie H., Yin C., Li Y., Wei H., Liu B., Tai Y., Fang J., Shao X., Jin X., Fang J, & Liu B. (2022). Electroacupuncture Ameliorates Mechanical Allodynia of a Rat Model of CRPS-I via Suppressing NLRP3 Inflammasome Activation in Spinal Cord Dorsal Horn Neurons. Frontiers in Cellular Neuroscience, 16, 826777.

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Immunofluorescence Staining of Brain Slices

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Immunofluorescence staining was performed as previously described [36 (link)]. Briefly, after being blocked with 5% donkey serum in 0.3% Triton X-100 for 60 min at room temperature, the brain slices were incubated at 4°C overnight with the following primary antibodies: anti-CB1R (1: 100, ab259323, Abcam, MA, USA), anti-NeuN (1: 1000, ab104224, Abcam, MA, USA), anti-GFAP (1: 50, ab4648, Abcam, MA, USA), anti-Iba1 (1: 100, ab5076, Abcam, MA, USA), anti-LC3B (1: 1000, ab192890, Abcam, MA, USA), and anti-TOMM20 (1: 100, ab56783, Abcam, MA, USA). Then, the slices were incubated with the appropriate fluorescence-conjugated secondary antibody (1: 500, Abcam, MA, USA) at 37° C for 1 h.

Liu B., Tian Y., Li Y., Wu P., Zhang Y., Zheng J, & Shi H. (2022). ACEA Attenuates Oxidative Stress by Promoting Mitophagy via CB1R/Nrf1/PINK1 Pathway after Subarachnoid Hemorrhage in Rats. Oxidative Medicine and Cellular Longevity, 2022, 1024279.

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Triple-Staining for Neurogenesis Quantification

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A triple-staining procedure was implemented for BrdU/DCX/NeuN to allow analyzation through immunofluorescence. The brain sections were incubated overnight at 4°C with the following primary antibodies: rat anti-BrdU (1:200, ab6326, Abcam), mouse anti-NeuN (1:500, ab104224, Abcam) and rabbit anti-DCX (1:500, ab18723, Abcam). The sections were washed in PBS and incubated for 4 h with fluorescent secondary antibodies: Alexa Fluor 488 goat anti-rat IgG to reveal immunoreactivity of BrdU, Alexa Fluor 405 donkey anti-mouse IgG to reveal immunoreactivity of NeuN, and Alexa Fluor 594 goat anti-rabbit IgG to reveal immunoreactivity of DCX, respectively (1:400 for all three antibodies, Abcam). Every sixth coronal section from the adult dentate gyrus was used to assess BrdU+/NeuN+/DCX- and BrdU+/NeuN+/DCX+ positive cells in both hemispheres (10 sections per animal, 3 mice per group). BrdU+/NeuN+/DCX- and BrdU+/NeuN+/DCX+ positive cells were observed and counted under an Olympus BX63 microscope fitted with a ×10 eyepiece lens with a ×40 objective, and their percent distribution in the BrdU positive cells was calculated.

Zhao F., Tao W., Shang Z., Zhang W., Ruan J., Zhang C., Zhou L., Aiello H., Lai H, & Qu R. (2020). Facilitating Granule Cell Survival and Maturation in Dentate Gyrus With Baicalin for Antidepressant Therapeutics. Frontiers in Pharmacology, 11, 556845.

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