Samples were fixed with 4% PFA for 10 min at room temperature, subsequently washed with PBS, permeabilized, and blocked in blocking solution (with 2% BSA, 0.1% Triton-X100 in PBS) for 2 h. Primary antibodies were added at 1:100 dilution [rabbit polyclonal anti-MyoVIIa (Proteus); mouse monoclonal anti-Sox2 (Millipore); rabbit polyclonal anti-Sox2 (Invitrogen); rat anti-E-cadherin (Abcam); mouse anti-GATA3 (Thermo Fisher Scientific); mouse anti-Islet 1 (DSHB, deposited by Jessell T.M.); goat anti-Doublecortin (Santa Cruz Biotechnology); rabbit anti-Pax2 (Thermo Fisher Scientific); rabbit anti-Pax8 (Abcam); mouse anti-Nestin (BD Transduction Laboratories); mouse anti-βIII-Tubulin (R&D); rabbit anti-Peripherin (Millipore); and mouse monoclonal anti-Brn3a (Millipore)], and incubated in blocking solution overnight at 4°C. Samples were then washed three times with PBS, followed by the addition of Alexa Fluor conjugated secondary antibodies (Invitrogen) at 1:500 dilution in blocking buffer for 2 days at room temperature. The images were acquired with a confocal microscope (Zeiss LSM 700) using 10× and 20× air objectives.
Rabbit anti pax2
Manufactured by Thermo Fisher Scientific
Sourced in United States
Rabbit anti-Pax2 is a primary antibody that specifically binds to the Pax2 protein. Pax2 is a transcription factor involved in the regulation of gene expression during development. This antibody can be used to detect and study the Pax2 protein in various research applications.
Automatically generated - may contain errors
Lab products found in correlation
Mouse anti neun, by Merck Group (2 mentions)
Lsm 700, by Zeiss (1 mentions)
Goat anti doublecortin, by Santa Cruz Biotechnology (1 mentions)
Alexa fluor conjugated secondary antibody, by Thermo Fisher Scientific (1 mentions)
Mouse monoclonal anti sox2, by Merck Group (1 mentions)
Mouse anti βiii tubulin, by R&D Systems (1 mentions)
Rat anti e cadherin, by Abcam (1 mentions)
Mouse anti nestin, by BD (1 mentions)
Mouse monoclonal anti brn3a, by Merck Group (1 mentions)
Rabbit anti peripherin, by Merck Group (1 mentions)
Triton x 100, by Merck Group (1 mentions)
Alexa fluor 647, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 555, by Thermo Fisher Scientific (1 mentions)
Gfp 1020, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (1 mentions)
Ecl chemiluminescence reagent, by Thermo Fisher Scientific (1 mentions)
Bca protein assay, by Beyotime (1 mentions)
Protease inhibitor phenylmethanesulfonyl fluoride, by Beyotime (1 mentions)
Ripa buffer, by Beyotime (1 mentions)
Anti gapdh antibody, by Abcam (1 mentions)
10 protocols using rabbit anti pax2
1
Immunofluorescence Staining of Cell Samples
2
Immunohistochemical Profiling of Murine Spinal Cord
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Young adult mice were deeply anaesthetized and fixed by perfusion with 4% paraformaldehyde. The spinal cord was dissected and post-fixed in 4% paraformaldehyde overnight at 4°C then rinsed thoroughly with 1X phosphate buffer saline (PBS). Transverse sections 60 µm thick were cut with a vibrating microtome from mid-lumbar spinal cord segments (L2 or L3) and processed free-floating for immunocytochemistry. Sections were washed in 50% ethanol for 30 min, rinsed 4 × 5 min in PBS containing 0.3% Triton X-100 (Sigma, St. Louis MI), and incubated in primary antibodies for three nights at 4°C. These were revealed with species-specific secondary antibodies raised in donkey and conjugated to Alexa fluor 488, Alexa fluor 555, or Alexa fluor 647 (Life Technologies, Carlsbad, CA) diluted 1:500 in and incubated overnight at 4°C. Sections were scanned with a confocal microscope (Nikon AR1) through a 20x or 60x oil-immersion. Scans were analyzed with NeNIS Elements Software (Nikon, Melville NY). Primary antibodies used were chicken anti-green fluorescent protein (Aves Labs, Tigard OR, GFP-1020, 1:2000), rabbit-anti Pax2 (ThermoFisher Scientific, Waltham MA, 71–6000, 1:1000), mouse anti-NeuN (Millipore, Billerica MA, MAB377, 1:1000), and rabbit-anti Nos1 (ThermoFisher Scientific, 61–7000, 1:500).
3
Western Blot Analysis of PAX2, CDK1 and GAPDH
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The cells were lysed with RIPA buffer (Beyotime, shanghai, China) and 1 mM protease inhibitor phenylmethanesulfonyl fluoride (Beyotime, shanghai, China), agitated for 15 minutes at 4℃, and centrifuged at 12,000 rpm for 25 minutes. Protein concentrations were measured with the BCA Protein Assay (Beyotime, Shanghai, China). Proteins were electrophoresed on SDS-polyacrylamide gels and transferred onto a polyvinylidene fluoride for analysis. Then, the membrane was incubated with rabbit anti-PAX2 (1:200 dilutions; Invitrogen, Carlsbad, CA), anti-CDK1(1:2000 dilutions; Abcam, San Francisco, USA) and anti-GAPDH antibody(1:5000 dilution; Abcam, San Francisco, USA) overnight at 4℃, respectively, and then incubated in the goat anti-rabbit IgG conjugated with horseradish peroxidase (1:5000 dilution; MT-bio, shanghai, China) at room temperature for 1 hour. Antibody binding was visualized using ECL chemiluminescence reagent (Thermo, MA, USA). GAPDH was used as an internal control to normalize other proteins expression level.
4
Immunofluorescence Staining of Mouse Brain Tissue
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Mice were deeply anesthetized with isoflurane and perfused intracardially with 4% paraformaldehyde in PBS64 . Tissues were dissected, post-fixed for 8 h, and cryoprotected in 20% sucrose overnight at 4 °C. Free-floating frozen sections were blocked in a 0.01 M PBS containing 2% donkey serum and 0.1% Triton X-100 followed by incubation with primary antibodies overnight at 4 °C and secondary antibodies for 2 h at room temperature. Sections were mounted with FluoromountG (Southern Biotech). The following primary antibodies were used: chicken anti-GFP (1:500, Aves Labs, GFP-1020), rabbit anti-c-Fos (1:4000, Abcam, ab190289), rabbit anti-lmx1b (1:500, Invitrogen, Cat.PA5-34471), rabbit anti-Pax2 (1:500, Invitrogen, Cat.71-6000) and rabbit anti-NKB (1:500, Novus, Cat.NB300-201). The following secondary antibodies were used: Alexa-Fluor 488 conjugated donkey anti-chicken (1:500, Jackson ImmunoResearch, 703-545-155), Cy3-conjugated donkey anti-rabbit (1:500, Jackson ImmunoResearch, 711-165-152) and 488-conjugated donkey anti-rabbit (1:500, Jackson ImmunoResearch, 711-545-152). Fluorescent Images were taken using a Nikon C2+ confocal microscope system (Nikon Instruments, Inc.).
5
Spinal Cord Development: Imaging Molecular Markers
6
Immunostaining of Islet1 and Pax2 in Tissue Sections
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Sections were washed with 0.1% Triton X-100 in 1X PBS (PBST), blocked with 1X PBS containing 10% lamb serum, 1% bovine serum albumin, and 0.25% Triton X-100 for 90 min and incubated with the appropriate primary antibodies diluted in blocking solution at 4 °C overnight. The following day, sections are washed briefly with PBST and incubated with the appropriate fluorescently-conjugated secondary antibodies diluted in blocking solution at 4 °C overnight. The following primary and secondary antibodies were used at the given dilution: Mouse monoclonal mouse anti-Islet1 (1:100, 39.45D-DSHB), rabbit anti-PAX2 (1 mg/mL, Invitrogen), goat antirabbit AlexaFluor 488 (1:500; Invitrogen), and goat antimouse AlexaFluor 568 (1:500; Invitrogen, Carlsbad, CA, USA). Stained sections were dehydrated in serial dilutions of ethanol in 1X PBS and mounted using DPX mounting media.
7
Antibody Validation for Protein Detection
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α-Vax1 and α-Vax2 were produced as reported previously (Mui et al., 2005 (link)). Commercially available antibodies against the following proteins were used: mouse anti-Myc (Santa Cruz Biotechnology, Dallas, TX, USA), mouse anti-GFP (Santa Cruz Biotechnology, Dallas, TX, USA), mouse anti-tubulin β-III (Tuj1; Covance, Princeton, NJ, USA), goat anti-Sox2 (Santa Cruz Biotechnology, Dallas, TX, USA), mouse anti-Nestin (RC2; Millipore, Billerica, MA, USA), mouse anti-β-galactosidase (Developmental Studies Hybridoma Bank, DSHB), mouse anti-NF160 (Developmental Studies Hybridoma Bank [DSHB], Iowa City, IA, USA), goat anti-Sdc2 (Santa Cruz Biotechnology, Dallas, TX, USA), goat anti-Sdc3 (Santa Cruz Biotechnology (Dallas, TX, USA), for immunohistochemistry), rabbit anti-Sdc3 (Abcam (UK), for Western blot), rabbit anti-Glp1 (Santa Cruz Biotechnology, Dallas, TX, USA), and rabbit anti-Pax2 (Invitrogen, Carlsbad, CA, USA) antibodies.
8
Embryonic Development Immunohistochemistry Protocol
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Embryos were harvested at E14.5, fixed overnight in 4% paraformaldehyde at 4°C, washed in PBS, dehydrated through a graded ethanol series, and stored in 100% ethanol at −20°C. Embryos were rehydrated in PBS, cryoprotected in 30% sucrose overnight at 4°C, embedded in Tissue-Tek OCT Compound (Sakura Finetek USA, Inc., Torrance, CA), quick-frozen on dry ice, and cryosectioned at 16 µm. Primary antibodies used for immunohistochemistry and their dilutions are as follows: mouse anti-neurofilament (1:250, 2H3), mouse anti-islet1/2 (1:100, 39.4D5), mouse anti AP-2alpha (1:100, 5E4) were obtained from Developmental Studies Hybridoma Bank (University of Iowa, Iowa City, IA); rabbit anti-Pax2 (1:250, 71-6000, Invitrogen); mouse anti-Ki67 (1:1000, ACK02, Leica Biosystems). Detection of primary antibodies was achieved using secondary antibodies conjugated to Cy3 (1:100, 115-106-006, Jackson ImmunoResearch Laboratories) or Alexa 488 (1:100, A32723, Molecular Probes). Section in situ hybridization was performed with digoxygenin-UTP-labeled riboprobes essentially as described (Nissim et al., 2007 (link)). At least three to five embryos in the experimental and control groups were evaluated for each antibody or in situ probe.
9
Comprehensive Immunostaining of Brain Tissue
10
Antibody Usage in Neural Research
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Antibodies used in this study were chick anti-bgal (RRID: AB_307210; Abcam), chick anti-GFP (RRID: AB_11180610; Invitrogen), guinea pig anti-Lbx1 (RRID: AB_2532144; Mu ¨ller et al., 2002) , guinea pig anti-Tlx3 (RRID: AB_2532145; Mu ¨ller et al., 2005) , guinea pig anti-vGlut1 (RRID: AB_2301751; Chemicon), goat anti-ChAT (RRID: AB_2079751; Chemicon), goat anti-Choleratoxin B (RRID: AB_211712; Calbiochem), rabbit anti-Pax2 (RRID: AB_88410; Invitrogen), rabbit anti-RFP (RRID: AB_2209751; Rockland), and sheep anti-GFP (RRID: AB_619712; Biogenesis). Fluorophore-coupled secondary antibodies used in this study were purchased from Jackson or Invitrogen. For detection of PSAM-GlyR, we used Alexa Fluor 488-conjugated aBungarotoxin (Invitrogen).
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