Immunoblots were performed as described [11 ] using the following antibodies: rabbit anti-Nurr1 (1:500; Santa Cruz, Dallas, TX), anti-Rabbit HRP (1:5,000; Cell Signaling), mouse anti-Beta Actin (1:1,000; Sigma, St. Louis, MO) and anti-mouse HRP (1:5,000; Cell Signaling, Danvers, MA). For immunofluorescence staining, N2A cells and primary neurons fixed and stained as previously described [12 ]. Primary antibodies used: rabbit polyclonal anti-Nurr1 (1:250; Santa Cruz, Dallas, TX), chicken polyclonal anti-Tyrosine Hydroxylase (1:500; Abcam, Cambridge, MA), rabbit polyclonal anti-Flag (1:500; Sigma F-7425). Secondary antibodies used: Alexafluor647 (1:500; Invitrogen, Carlsbad, CA) and Alexafluor488 (1:500; Invitrogen). All imaging was performed as previously described [11 ]
Rabbit anti nurr1
Manufactured by Santa Cruz Biotechnology
Sourced in United Kingdom
Rabbit anti-Nurr1 is a primary antibody product manufactured by Santa Cruz Biotechnology. It is designed to detect the Nurr1 protein, which is a nuclear receptor transcription factor that plays a role in the development and maintenance of dopaminergic neurons.
Automatically generated - may contain errors
Lab products found in correlation
Rabbit anti mecp2, by Cell Signaling Technology (2 mentions)
Alexa fluor 647, by Thermo Fisher Scientific (1 mentions)
Anti flag, by Merck Group (1 mentions)
Anti tyrosine hydroxylase, by Abcam (1 mentions)
Mouse anti β actin, by Merck Group (1 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (1 mentions)
Anti nurr1, by Santa Cruz Biotechnology (1 mentions)
Abc kit, by Vector Laboratories (1 mentions)
Rabbit anti tyrosine hydroxylase th, by Merck Group (1 mentions)
Biotinylated goat anti rabbit igg antibody, by Vector Laboratories (1 mentions)
Rabbit anti gdnf, by Santa Cruz Biotechnology (1 mentions)
Axio observer z1, by Zeiss (1 mentions)
Mouse anti tuj1, by Promega (1 mentions)
Chicken anti tuj1, by Merck Group (1 mentions)
Goat anti chl1, by R&D Systems (1 mentions)
Chicken anti th, by Abcam (1 mentions)
Alexa fluor 488 or 555, by Thermo Fisher Scientific (1 mentions)
Mouse anti map2, by Merck Group (1 mentions)
Rat anti dat, by Merck Group (1 mentions)
Mouse anti tuj1, by Fortrea (1 mentions)
8 protocols using rabbit anti nurr1
1
Nurr1 Protein Detection and Localization
2
Immunohistochemical Analysis of Dopaminergic Neurons
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Animals were sacrificed via pentobarbital overdose (60 mg/kg) and intracardially perfused with saline, then 4% paraformaldehyde. Brains were removed and post-fixed in 4% paraformaldehyde for 24 h and then dehydrated in 30% sucrose. The sections at a thickness of 16 μm were cut through the substantia nigra (−2.92 to −3.88 mm from bregma) (Paxinos and Watson, 2007 ). The tissue sections were incubated in 0.3% H2O2 for 45 min, and blocked in 10% goat serum for 1 h. The sections were incubated overnight at 4°C with rabbit anti-tyrosine hydroxylase (TH; 1:300; Millipore), rabbit anti-dopamine transporter (DAT; 1:200; Santa Cruz), rabbit anti-GDNF (1:200; Santa Cruz Biotechnology) or rabbit anti-Nurr1 (1:100; Santa Cruz Biotechnology). Subsequently, the sections were incubated with biotinylated goat anti-rabbit IgG antibody (1:250; Vector labs) and avidin-biotin complex (ABC-kit, Vector Labs) at 37°C for 30 min, and then stained with 3,3′-diaminobenzidine (DAB). Then the sections were observed under a light microscope (BX51; Olympus, Tokyo, Japan). Image scanning analysis system (Image-Pro Plus) was used to analyze the changes in integrated optical density (IOD) of DAT, TH and GFAP.
3
Immunohistochemistry of Developmental Embryos
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Embryos were fixed in 4% paraformaldehyde (2–8 hrs, dependent on developmental age), cryopreserved in 20% sucrose and coronally sectioned (16 μm, 1:10 series). For primary cultures and explant assays, cells were fixed with 4% paraformaldehyde, rinsed and stored in PBS with 0.03% sodium azide.
Immunohistochemistry was performed using previously described methods21 (link). Antibodies used were as follows: goat anti-CHL1 (1:500, R&D Systems), rabbit anti-NURR1 (1:200, Santa Cruz), rabbit anti-TH (1:800, PelFreez), chicken anti-TH (1:400, Abcam), mouse anti-TUJ1 (1:15000, Promega), and chicken anti-TUJ1 (1:200, Millipore). Appropriate secondary antibodies, donkey 488, 555 and 647 Alexaflour (Jackson ImmunoResearch Laboratories), were used at a dilution of 1:400. Images were captured using fluorescence microscope (Zeiss Axio Observer Z1 or Zeiss 780 confocal microscrope).
Immunohistochemistry was performed using previously described methods21 (link). Antibodies used were as follows: goat anti-CHL1 (1:500, R&D Systems), rabbit anti-NURR1 (1:200, Santa Cruz), rabbit anti-TH (1:800, PelFreez), chicken anti-TH (1:400, Abcam), mouse anti-TUJ1 (1:15000, Promega), and chicken anti-TUJ1 (1:200, Millipore). Appropriate secondary antibodies, donkey 488, 555 and 647 Alexaflour (Jackson ImmunoResearch Laboratories), were used at a dilution of 1:400. Images were captured using fluorescence microscope (Zeiss Axio Observer Z1 or Zeiss 780 confocal microscrope).
4
Immunofluorescence Staining of Neuronal Markers
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Cells were fixed with 4% paraformaldehyde for 5 min and permeabilized with 0.2% Triton X-100/PBS. After blocking (4% BSA/1% Cosmic Calf Serum in PBS) for at least 30 min, primary antibodies were added and incubated overnight at 4°C. After three washes, secondary antibody conjugated with Alexa Fluor 488 or 555 (Molecular Probes) was added and incubated for 60 min. The wells were then incubated with 4′,6-diamidino-2-phenylindole (DAPI) nuclear counterstain for 5 min before analysis. Antibodies used were sheep anti-TH (Pel-Freez), mouse anti-EN1 (Development Studies Hybridoma Bank, IA), rabbit anti-PRPH (Millipore), mouse anti-MAP2 (Sigma), mouse anti-TUJ1 (Covance), rabbit anti-TUJ1 (Covance), rabbit anti-PITX3 (Invitrogen), rabbit anti-NURR1 (Santa Cruz Biotechnologies), goat anti-FOXA2 (Santa Cruz), mouse anti-SYN1 (Synaptic Systems), and rat anti-DAT (Millipore). See also Table S2 .
5
Immunofluorescence Staining for Nurr1
6
Immunohistochemical Analysis of Hippocampal Proteins
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After the CPP test, four rats per group were randomly assigned to immunohistochemistry test. Rats were deeply anesthetized using 3% sodium pentobarbital i.p (30 mg/kg) and perfused with 4% paraformaldehyde (PFA) in 0.1 M phosphate buffer (pH 7.4) intracardially. The brains were then removed and postfixed in 4% PFA immediately. After dehydration and paraffin embedding, the hippocampus was cut into 5-μm sections using a Leica RM2135 microtome. Immunohistochemistry was performed as described in product manual of streptavidin-biotin complex with peroxidase (Rabbit IgG) Kit (Boster, China). Antibodies for rabbit anti-p-CREB (1:150 dilution; Abcam, UK), rabbit anti-Nurr1 (1:50 dilution; Santa Cruz, USA), and rabbit anti-BDNF (1:150 dilution; Abcam, UK) were used to determine the levels of p-CREB, Nurr1, and BDNF. Five slices per rat containing hippocampus were selected, and then, three horizons of each slice were randomly observed in the light microscope (×40). Positive expression of p-CREB, Nurr1, and BDNF was defined as appearance of brown particles within the cell. We used Image-pro plus software (IPP 6.0, Media Cybernetics Inc., Silver Spring, Maryland, USA) to measure positive cells’ integrated optical density, and the relative content for p-CREB, Nurr1, and BDNF was represented by the mean value of each group.
7
Immunofluorescence Staining of Midbrain Cells
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Cells were fixed with 4% paraformaldehyde for 20 minutes, permeabilized and blocked with phosphate buffered saline with 0.3% Triton-X100 and 1% bovine serum albumin for 40 minutes, then incubated with first antibodies diluted with blocking solution at 4℃ overnight. Alexa Fluo series of second antibodies (Thermo Scientific)
were applied accordingly for two hours at room temperature. Cells were finally mounted in 4',6-diamidino-2-phenylindole (DAPI) and examined using fluorescence microscope (Leica DMi8). Embryonic and postnatal ventral midbrain tissues were fixed with 4% paraformaldehyde and dehydrated with 30% sucrose overnight, and cryosectioned at 14 μm thickness. The first antibodies used include rabbit anti-MeCP2
(Cell Signaling Technology), mouse anti-NURR1 (R&D Systems), mouse anti-TH (Sigma), rabbit anti-NURR1 (Santa Cruz Biotechnology).
were applied accordingly for two hours at room temperature. Cells were finally mounted in 4',6-diamidino-2-phenylindole (DAPI) and examined using fluorescence microscope (Leica DMi8). Embryonic and postnatal ventral midbrain tissues were fixed with 4% paraformaldehyde and dehydrated with 30% sucrose overnight, and cryosectioned at 14 μm thickness. The first antibodies used include rabbit anti-MeCP2
(Cell Signaling Technology), mouse anti-NURR1 (R&D Systems), mouse anti-TH (Sigma), rabbit anti-NURR1 (Santa Cruz Biotechnology).
8
Chromatin Immunoprecipitation and Methylation Analysis
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For ChIP experiments, cells were cross-linked with 1% paraformaldehyde for 15 minutes and Chromatins were sheared into an average 200-400 bp in length by sonication (Diagenode) and immunoprecipitated with following antibodies: rabbit anti-MeCP2 (Cell Signaling Technology), rabbit anti-NURR1 (Santa Cruz Biotechnology) and rabbit anti-TET1 (Abcam). Immunoprecipitated DNA fragments were collected by magnetic beads (Active Motif), purified, and subjected to real-time PCR using primers specific to regions spanning three NBREs on Th promoter. Data were normalized to values of the input DNA. For MeDIP and hMeDIP experiments, genomic DNA were extracted from cells, sheared, immunoprecipitated, collected and subjected to real-time PCR in a similar way as ChIP with following antibodies: mouse anti-5mC (Abcam) and rabbit anti-5hmC (Active Motif). The primers are listed in Supplemental Table 1.
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