Brain slides were blocked with 1% BSA/PBS for 1 h at RT and incubated with anti-MBP (1:500, Abcam, Burlingame, CA, USA), anti-Iba-1 (1:300, BD Bioscience, USA), anti-NF-κB/p65 (1:200, Cell Signaling Technology, Danvers, MA, USA), anti-TLR4 (1:200, Bioworld Tech. Inc., St. Louis Park, MN, USA), anti-iNOS (1:300, Abcam, Burlingame, CA, USA), anti-Arg-1 (1:300, Gene Tex, Irvine, CA, USA), anti-GFAP (1:250, Thermo Fisher, USA), anti-BDNF (1:200, Novusbio, Centennial, CO, USA), anti-GDNF (1:500, Abcam, Burlingame, CA, USA), anti-NG2 (1:300, Millipore, Germany) and anti-Ki67 (1:200, BD Pharmingen, USA) at 4°C for overnight, followed by corresponding secondary antibodies at RT for 1 h. The results were repeated three times with consecutive brain slices from each group, and the slides were observed under fluorescence microscopy in a blinded fashion. Analysis and quantification were done in three sections/per mouse by Image-Pro Plus 6.0 software.
Anti gdnf
Manufactured by Abcam
Sourced in United States, United Kingdom
Anti-GDNF is a lab reagent used to detect and quantify the presence of Glial Cell Derived Neurotrophic Factor (GDNF) in samples. It is a tool for researchers to study GDNF, which is a protein involved in the survival and growth of various types of neurons.
Automatically generated - may contain errors
Lab products found in correlation
Anti ki67, by BD (3 mentions)
Anti nf κb p65, by Cell Signaling Technology (2 mentions)
Anti gfap, by Thermo Fisher Scientific (2 mentions)
Anti arg 1, by GeneTex (2 mentions)
Anti iba 1, by BD (2 mentions)
Anti bdnf, by Novus Biologicals (2 mentions)
Anti inos, by Abcam (2 mentions)
Anti β actin, by Abcam (2 mentions)
Anti ret, by Abcam (2 mentions)
Anti ng2, by Merck Group (1 mentions)
Anti mbp, by Abcam (1 mentions)
Horseradish peroxidase linked secondary antibody, by Abcam (1 mentions)
Pvdf membrane, by Roche (1 mentions)
Anti bmp2, by Abcam (1 mentions)
Anti pdgfrα, by Merck Group (1 mentions)
Supersignal r west pico chemiluminescent substrate, by Thermo Fisher Scientific (1 mentions)
Anti psd95, by Cell Signaling Technology (1 mentions)
Ipvh0010, by Merck Group (1 mentions)
Bca protein assay kit, by Beyotime (1 mentions)
Ge amersham imager 600, by GE Healthcare (1 mentions)
10 protocols using anti gdnf
1
Immunohistochemical Analysis of Neuroinflammation
2
Protein Expression Analysis in Tissues
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Approximately 50 mg samples were minced to small pieces using surgical blades and sonicated in a protein lysis buffer containing protease inhibitors (complete, Ultra, Mini, EDTA-free, EASYpack Roche, Germany). Protein concentrations were measured by the BCA method, and samples were adjusted to the same protein concentration, aliquoted and stored at −80°C. Equal amounts of total proteins from tissues were separated on SDS-polyacrylamide gels and electro-transferred to PVDF membranes (Roche Germany). The blots were incubated with following polyclonal antibodies overnight at 4°C: anti-BMP2 (1:1000, Abcam), anti-GDNF (1:1000, Abcam), and anti-pSmad1/5/8 (1:1500, Abcam). Blots were washed and incubated with horseradish peroxidase-linked secondary antibodies (1:2000, Abcam) for 2 h at room temperature and detected using ECL reagent (Millpore, MA, United States).
3
Immunofluorescence Analysis of Neural Markers
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Cells were blocked with 1% BSA/PBS for 1 h at RT and incubated with anti-Iba-1 (1:300, BD Bioscience, San Jose, CA, USA), anti-NF-κB/p65 (1:200, Cell Signaling Technology, USA), anti-iNOS (1:300, Abcam, Burlingame, CA, USA), anti-Arg-1 (1:300, Gene Tex, Irvine, CA, USA), anti-GFAP (1:250, Thermo Fisher, USA), anti-BDNF (1:200, Novusbio, Centennial, CO, USA), anti-GDNF (1:500, Abcam, USA), anti-PDGFRα (1:300, Millipore, Germany) and anti-Ki67 (1:200, BD Pharmingen, USA) at 4°C for overnight, followed by corresponding secondary antibodies at RT for 1 h. The results were repeated three times with similar results. Analysis and quantification were done by Image-Pro Plus 6.0 software.
4
Hippocampal Protein Expression Analysis
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At 24 h after DEX injection, bilateral hippocampi of P8 pups for GDNF/NCAM/CREB analysis (n=5 per group) and P35 pups for PSD95/GAP43 analysis (n=5 per group) were rapidly harvested, flash-frozen, and stored at −80°C until use. Frozen hippocampi were cut and then lysed on ice for 30 min. The lysate was centrifuged and total protein concentration measured using a BCA Protein Assay Kit (P0010; Beyotime, China). Equal amounts of protein per gel lane were separated by electrophoresis using 12.5% SDS-polyacrylamide gels and electrotransferred to polyvinylidene fluoride membranes (IPVH0010; Millipore, Germany). The membranes were blocked in 5% non-fat milk diluted in Tris-buffered saline with Triton X (TBST) for 1 h, incubated with the indicated primary antibodies overnight at 4°C, and then incubated in secondary antibodies for 2 h at room temperature. Protein bands were visualized and photographed using SuperSignal R West Pico Chemiluminescent Substrate (34080; Thermo, USA) and a GE Amersham Imager 600. The primary antibodies used were anti-GDNF (1:500 Ab18956; Abcam, Cambridge, UK), anti-NCAM (1:1000, 3606S; Cell Signaling Technology, Danvers, MA, USA), anti-PSD95 (1:1000, 3450S; Cell Signaling Technology), anti-GAP43 (1:1000, 8945S; Cell Signaling Technology), and anti-CREB (1:1000, 9197S; Cell Signaling Technology).
5
Culturing Human Pancreatic Cancer Cells
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The human PC cell lines (BxPC3 and Panc-1) were obtained from the Chinese Academy of Sciences Cell Bank of Type Culture Collection, and they were cultured in the corresponding medium (Invitrogen) which contains 10% fetal bovine serum (FBS), 100 μg/ml penicillin-streptomycin. The cells were incubated at 37°C in a humidified atmosphere containing 5% CO2. Antibodies were purchased from the following sources: recombinant mature human Nodal (rhNodal) (R&D Systems) and its inhibitor SB431542 (Sigma-Aldrich); anti-MMP-9 (Bioworld); and anti-NGF, anti-BDNF, and anti-GDNF (Abcam).
6
SDS-PAGE and Immunoblotting of Cell Proteins
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Total cell protein was separated on 10% SDS-PAGE (Pall Corporation, USA), and transfected to the membranes (EMD Millipore, USA). Then, the membranes were blocked with 5% BSA for 1 h and immunoblotted with anti-CCL2 (1:500, affinity, USA), anti-CCR2 (1:500, affinity, USA), anti-GDNF (1:500, Abcam, USA), anti-p-RET (1:500, affinity, USA), anti-RET (1:500, affinity, USA), or anti-β-actin (1:1,000, affinity, USA). The results were visualized with a chemiluminescence reagent (Thermo, USA).
7
Immunocytochemical Analysis of Sertoli Cells
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For immunocytochemical staining, the immortalized human Sertoli cells were fixed with 4% paraformaldehyde (PFA) for 15 min, washed three times with phosphate-buffered saline (PBS) for 5 min each and permeabilized in 0.5% triton-X 100 (Sigma) for 20 min. After washing with PBS, the cells were blocked in 3% serum or BSA for 60 min and followed by incubation with primary antibodies, including anti-SOX9 (Millipore), anti-WT1 (Santa Cruz), anti-OCLN (Abcam), anti-VIM (vimentin, Santa Cruz), anti-SCF (Sigma), anti-BMP4 (Abcam), anti-GDNF (Abcam), anti-3β-HSD (Santa Cruz), anti-VASA(Santa Cruz), and anti-Ki-67 (BD Biosciences), overnight at 4°C. Replacement of primary antibodies with isotype IgGs (Santa Cruz) served as negative controls. After washing three times in PBS for 10 min each, the cells were incubated with the secondary antibody (Sigma) conjugated with rhodamine at a 1:200 dilution for 1 hour at room temperature. DAPI (4, 6-diamidino-2-phenylindole, Sigma) was used to counterstain the nuclei, and images were captured with a fluorescence microscope (Nikon).
8
Quantifying Neurobiological Markers in Mouse Brain
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The mouse brain was extracted using protein extraction buffer (T-PER, Thermo Fisher, USA) containing protease and phosphatase inhibitors (Thermo Fisher, USA). Equal amounts of protein extracts (20µg) were separated by SDS-PAGE and transferred onto a nitrocellulose lter (NC) membrane (GE Healthcare Life Sciences, USA). After being blocked with 5% non-fat milk, membranes were incubated at 4°C overnight with primary antibodies (all at 1:1000) as follows: anti-β-actin (Abcam, USA), anti-p-mTOR(Abcam, USA), anti-GDNF(Abcam, USA), anti-BNDF(Abcam, USA), anti-CNTF(Abcam, USA), anti-Bcl-2(Abcam, USA), anti-Bax (Abcam, USA), anti-p110α-PI3K (C73F8) (Cell Signaling Technology, MA), anti-p-Akt (Ser473) (Cell Signaling Technology, MA), anti-β-catenin (Cell Signaling Technology, MA), anti-Fzd1 (R&D system, USA), anti-NLRP3 (R&D system, USA), anti-WNT1 (ABGENT, USA), anti-iNOS/NOS(BD, USA) or anti-Arginase1 (BD, USA). After being washed in TBST, the immunoblots underwent incubation with horseradish peroxidase-conjugated secondary antibodies (Jackson Lab, USA) for 1h. Bands were visualized and analyzed using a ChemiDoc XRS (Bio-Rad, USA) and Image Lab(Bio-Rad, USA).
9
Immunohistochemical Analysis of Murine Brain
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Anesthetized mice were transcardially perfused with cold paraformaldehyde 4 %. The brain was isolated and fixed in 4%PFA. Then the brains were embedded in paraffin. Coronal sections were cut at a 5-μm thickness with a microtome (Leica, Germany). Sections were incubated with primary Ab for 2h at 37°C: anti-TH 1:200 (Abcam, Cambridge, UK), GFAP 1:500 (Abcam, Cambridge, UK), Iba1 1:4000 (Abcam, Cambridge, UK), anti-GDNF 1:100 (Abcam, Cambridge, UK), Sections were then incubated with secondary Ab biotinconjugated goat anti-mouse (KPL) for 30min at 37°. Staining was visualized using a solution of 0.05 % 3,3'-diaminobenzidine (DAB), 50 mM Tris-HCl pH 7.2, 0.02 % H2O2. Images were captured with an Olympus BX51 microscope. Microglial and astroglia cells were analysed with ImageJ program in a double-blind design. The images were analysed for staining intensity which represents the level of target protein present. Four sections were randomly chosen through the midbrain, with a total of 12 sections per individual animal using unbiased stereological rules. The striatum and the substantia nigra was analysed as shown in the sample images. All analysis was done blind to treatment.
10
Western Blot Analysis of Protein Targets
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Approximate 2 3 10 6 cultured human CCs were collected for the following experiments. Total protein was extracted using the Radioimmunoprecipitation lysis buffer (Beyotime, Haimen, China) containing 1% 4-(2-aminoethyl) benzenesulfonyl fluoride hydrochloride. Proteins were separated by SDS-PAGE using a 4% stacking gel and a 10% separating gel before they were transferred onto the polyvinylidene fluoride membranes. After being blocked with Tris-buffered saline with 0.1% Tween 20 buffer containing 5% BSA, the membranes were incubated with specific primary antibodies (anti-GDNF, anti-GFRA1, anti-RET, and anti-glyceraldehyde 3-phosphate dehydrogenase obtained from Abcam, diluted at 1:400) at 4°C overnight. The membranes were then washed with Tris-buffered saline with 0.1% Tween 20 and were incubated with the corresponding horseradish peroxidaseconjugated secondary antibody (1:2000; Abcam, Cambridge, MA) prior to visualizing the immunoreactive bands using the enhanced chemiluminescence detection system (Thermo Fisher Scientific).
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