Localization of dCSF-CNs and the effect of targeted ablation of dCSF-CNs by icv administration of CB-SAP were assessed by double-staining immunofluorescence. Anti-CB (Abcam) and anti-NeuN (Santa Cruz Biotechnology) primary antibodies were used to treat sections of dCSF-CNs (4 μm) overnight at 4°C. The sections were then incubated with secondary antibodies for 1 h, washed, and visualized under a Zeiss microscope.
Anti neun
Manufactured by Santa Cruz Biotechnology
Sourced in United States
Anti-NeuN is a primary antibody that specifically binds to the NeuN (Neuronal Nuclei) protein, which is a widely used marker for identifying mature neurons. It can be used in various techniques, such as immunohistochemistry and immunocytochemistry, to visualize and quantify neuronal populations in tissue samples and cell cultures.
Automatically generated - may contain errors
Lab products found in correlation
Lsm 710 confocal microscope, by Zeiss (3 mentions)
Anti gfap, by Santa Cruz Biotechnology (2 mentions)
Anti gap43, by Abcam (1 mentions)
Anti nestin, by Abcam (1 mentions)
Thapsigargin tg, by Merck Group (1 mentions)
Anti beclin1, by Abcam (1 mentions)
Anti gapdh, by Santa Cruz Biotechnology (1 mentions)
Anti p62, by Santa Cruz Biotechnology (1 mentions)
Goat anti rabbit and anti mouse igg hrp, by Santa Cruz Biotechnology (1 mentions)
Rapamycin rapa, by Cell Signaling Technology (1 mentions)
Anti bfgf, by Santa Cruz Biotechnology (1 mentions)
3 methyladenine 3 ma, by Merck Group (1 mentions)
Recombinant human bfgf, by Merck Group (1 mentions)
Dulbecco s modified eagle s medium dmem, by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Anti lc3, by Abcam (1 mentions)
Anti s100β, by Merck Group (1 mentions)
Alexa fluor 488 and 594 conjugated secondary antibody, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 568, by Thermo Fisher Scientific (1 mentions)
Axiovert 200m fluorescent microscope, by Zeiss (1 mentions)
5 protocols using anti neun
1
Localization and Targeted Ablation of dCSF-CNs
2
Autophagy Regulation in Neural Cells
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All reagents we used were commercially available. Fetal bovine serum (FBS) and Dulbecco's modified Eagle's medium (DMEM) were purchased from Invitrogen (Carlsbad, California). Recombinant human bFGF was purchased from Sigma (Sigma‐Aldrich, St. Louis, Missouri). Anti‐GFAP, anti‐bFGF, anti‐p62, anti‐NeuN, and anti‐GAPDH antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, California). Anti‐GAP43, anti‐LC3, anti‐Beclin‐1, and anti‐Nestin antibodies were purchased from Abcam (CB, United Kingdom). Goat anti‐rabbit and anti‐mouse IgG‐HRP, goat anti‐chicken IgY H&L, donkey anti‐goat IgG H&L were purchased from Santa Cruz Biotechnology. An enhanced chemiluminescence kit and CM‐DiI were purchased from Bio‐Rad (Hercules, California). Thapsigargin (TG) and 3‐methyladenine (3‐MA) were purchased from Sigma‐Aldrich. The autophagy activator rapamycin (RAPA) was purchased from Cell Signaling Technology. All other reagents were purchased from Beyotime Institute of Biotechnology (Shanghai, China) unless otherwise specified.
3
Immunostaining of Neurological Markers
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Prepared samples were post-fixed with 4% PFA. After one washing with PBS and another washing with PBS including 0.3% Triton X-100 (PBS-T), followed by being blocked in blocking solution (5% bovine serum albumin and 5% fetal bovine serum in PBS-T) at room temperature (RT), samples were incubated with primary antibodies diluted in blocking solution overnight at 4 °C. The following primary antibodies were used: anti-AIMP1, anti-AIMP2, anti-AIMP3, anti-NeuN (Santa Cruz Biotechnology, CA, USA), anti-Neurofilament (NF) H&M (Millipore, Bedford, MA, USA), anti-p75 neurotrophin receptor (NTR), and anti-S100β (Sigma, St. Louis, MO, USA). Samples then were washed three times with PBS and then incubated with Alexa Fluor 488- and 594-conjugated secondary antibodies (Invitrogen, Carlsbad, CA, USA) in blocking solution for 2 hours at RT, washed three times with PBS, and mounted with cover glass.
4
Immunohistochemical Profiling of Neural Markers
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Sections were penetrated with 0.1% Triton X-100 (2 × 10 min at 25°C); washed in PBS (3 × 5 min at 25°C); blocked with 10% goat or donkey serum (30 min at 25°C); treated with 1:250 anti-IKKβ (Millipore), 1:500 anti-NeuN (Santa Cruz Biotechnology), 1:300 anti-GFAP (Santa Cruz Biotechnology), 1:1000 anti-IBA1 (Dako), and 1:1000 anti-GFP (Santa Cruz Biotechnology) antibodies (4°C overnight); washed in PBS (3 × 10 min at 25°C); and then subjected to reaction with fluorescence-conjugated secondary antibodies of 1:1000 Alexa Fluor 488 and 1:1000 Alexa Fluor 568 (Invitrogen; 2 h at 25°C); and rinsed with PBS (3 × 10 min at 25°C). The sections were mounted on slides using sterile 0.2% gelatin and DAPI mounting media (Vector Laboratories) and coverslipped. Images were taken using either an Axiovert 200M Fluorescent Microscope (Zeiss) equipped with a 20× objective or an LSM 710 Confocal Microscope (Zeiss) equipped with a 63× objective. For the immunohistochemistry, the following two sets of control experiments were performed to test specificity: (1) replacement of the primary antibody with only the serum of the appropriate species; and (2) omission of secondary antibodies. No immunostaining was detected under either of these conditions.
5
Western Blot Analysis of Neural Markers
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The NSCs were homogenized in lysis buffer and centrifuged (12000 g at 4℃) for 15 min. Equal amounts of protein (30 µg) from the supernatants were separated on a 10% acrylamide gel, and the proteins were electrotransferred onto nitrocellulose membranes. After blocking, the membranes were incubated with primary antibody at 4℃. The primary antibodies included anti-DCX (1:2000, Millipore, Billerica, MA, USA), anti-NeuN (1:1000, Santa Cruz Biotechnology, Santa Cruz, CA, USA), and anti-β-actin (1:2000, Santa Cruz Biotechnology) antibodies. The membranes were then incubated with secondary antibodies. The blots were rinsed, and protein bands were visualized using an enhanced chemiluminescence detection system (Amersham, Pittsburgh, PA, USA).
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