Hematoxylin and eosin (H&E) staining was performed on tissue sections, and light microscopic evaluation for pathological abnormalities was performed with the examiner blind to genotype. Standard immunohistochemical procedures were used to immunostain serial sections of tissues with antibodies for IF proteins and to counterstain with hematoxylin. Analyses of IF aggregates in paravertebral sympathetic-chain ganglia were performed using rabbit anti-peripherin (Abcam: ab4666; 1:2500), rabbit anti-vimentin (Abcam, Ab92547; 1:250), rabbit anti-alpha-internexin (Abcam, Ab40758; 1:250) and mouse anti-NF-heavy chain, hypophosphorylated (Covance, SMI-32R; 1:5000). Rabbit anti-peripherin (Abcam: ab4666; 1:1000) was used to stain IF aggregates in the enteric nervous system. Images were captured at 60x and 100x with oil immersion using the Olympus BX61 microscope (Olympus America), the QImaging Retiga 4000R camera (Surrey) and Volocity 6.2.1 software (Perkin Elmer, Inc.). Figures were created using Adobe Photoshop CS4.
Ab4666
Manufactured by Abcam
Sourced in United Kingdom, United States
Ab4666 is a type of lab equipment. It is a device used for a specific function in laboratory settings. The core function of Ab4666 is to perform a particular task required in scientific research and analysis. No further details about the intended use or specific applications of this product are provided.
Automatically generated - may contain errors
Lab products found in correlation
Ab13970, by Thermo Fisher Scientific (2 mentions)
Mab1195, by R&D Systems (2 mentions)
Ab5945, by Abcam (2 mentions)
Pa5 85263, by Thermo Fisher Scientific (2 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Ab178400, by Abcam (1 mentions)
Sox10, by Cell Signaling Technology (1 mentions)
Brn3a, by Merck Group (1 mentions)
Dako mounting medium, by Agilent Technologies (1 mentions)
Evos fl auto 2 imaging system, by Thermo Fisher Scientific (1 mentions)
Alexa fluor secondary antibody, by Thermo Fisher Scientific (1 mentions)
Ab53554, by Abcam (1 mentions)
Ab110987, by Abcam (1 mentions)
Anti tuj1 antibody, by Fortrea (1 mentions)
Ab70521, by Abcam (1 mentions)
Ab22736, by Abcam (1 mentions)
Anti ha, by Abcam (1 mentions)
Anti vinculin, by Santa Cruz Biotechnology (1 mentions)
Anti egfr, by Biosynth (1 mentions)
Ab7349, by Abcam (1 mentions)
11 protocols using ab4666
1
Immunohistochemical Analysis of Neuronal Proteins
2
Immunostaining of Differentiated Sensory Neurons
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After differentiation into sensory neurons on glass coverslips, cells were washed with PBS and fixed in 4% PFA for 15 min. After washing and permeabilization with 0.2% Triton X-100 for 1 h, coverslips were blocked with 10% BSA for 1 h at RT before incubation with primary antibodies at 4°C overnight. The following primary antibodies were used: Sox10 (#89356, Cell Signalling) at 1:1000 dilution; NEUROG1 (#mab3524, R&D) at 1:500 dilution, BRN3A.
(mab1585, Milipore) at 1:200, ISL1 (AB178400, Abcam) at 1:100 dilution, Tuj1 (MAB1637, Millipore) at 1:500 dilution, PRPH (AB4666, Abcam) at 1:100. After washing, coverslips were exposed to the required Alexa Fluor secondary antibodies (either Thermo Fisher Scientific or Abcam) and were incubated for 1 h at room temperature in the dark. Cultures were counterstained with DAPI and mounted onto slides using Dako mounting medium (Agilent). Images were captured using an EVOS FL Auto2 imaging system (Thermo Fisher) and processed with Fiji.45 (link)
(mab1585, Milipore) at 1:200, ISL1 (AB178400, Abcam) at 1:100 dilution, Tuj1 (MAB1637, Millipore) at 1:500 dilution, PRPH (AB4666, Abcam) at 1:100. After washing, coverslips were exposed to the required Alexa Fluor secondary antibodies (either Thermo Fisher Scientific or Abcam) and were incubated for 1 h at room temperature in the dark. Cultures were counterstained with DAPI and mounted onto slides using Dako mounting medium (Agilent). Images were captured using an EVOS FL Auto2 imaging system (Thermo Fisher) and processed with Fiji.45 (link)
3
Immunostaining of Human Neurons and DRG
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Human cells grown on 13-mm or 15-mm borosilicate coverslips pretreated with Matrigel were fixed in 4% paraformaldehyde for 15 minutes at room temperature and washed twice with PBS. The cells were incubated with primary antibodies for 24 hours at room temperature (RT). The cells were then washed with PBS and incubated with secondary antibodies at RT for 2 hours. The following antibodies were used for immunostaining: mouse anti-TuJ1 (1:100, R&D MAB1195), rabbit anti-Brn3a (1:500, Millipore #AB5945), rabbit anti-Peripherin (1:2000, Abcam, AB4666), guinea pig anti-TRPV1 (1:2000, gift from David Julius), rabbit anti-pSMAD1/5 (1:800, Cell Signaling #9516), and fluorophore-coupled secondary antibodies (1:1000, Alexa Fluor 488, 555, 594, 647, Thermo Fisher Scientific). Images were captured with a Carl Zeiss LSM 700 microscope and processed with Fiji/ImageJ (NIH).
For mouse DRG staining, mice were anesthetized with 2.5% Avertin and perfused transcardially with 1X PBS followed by 4% formaldehyde. Dissected DRG were first postfixed in the same fixative for 3 hours and stored overnight in 30% sucrose in PBS at 4°C before cryostat sectioning. The following primary antibodies were used for immunostaining: chicken anti-GFP (1:2000, Abcam #AB13970) and rabbit anti-ACVR1 (1:1000, Invitrogen #PA5-85263).
For mouse DRG staining, mice were anesthetized with 2.5% Avertin and perfused transcardially with 1X PBS followed by 4% formaldehyde. Dissected DRG were first postfixed in the same fixative for 3 hours and stored overnight in 30% sucrose in PBS at 4°C before cryostat sectioning. The following primary antibodies were used for immunostaining: chicken anti-GFP (1:2000, Abcam #AB13970) and rabbit anti-ACVR1 (1:1000, Invitrogen #PA5-85263).
4
Immunohistochemical Analysis of SIK3 Expression
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The following primary antibodies were used for immunohistochemistry experiments: primary Anti-SIK3 antibody (ab110987, Abcam, Cambridge, UK, and as a control: LS-c120369 SIK3 Lifespan Bioscience, Seattle, USA) at 1 : 50 (P0, P5) or 1 : 40 (4 weeks) concentration; anti-peripherin antibody (ab4666, Abcam, Cambridge, UK) at a 1 : 100 concentration; anti-TUJ1 antibody (Covance, New Jersey, US) at a 1 : 100 concentration; anti-GFAP antibody (ab53554, Abcam, Cambridge, UK) at a 1 : 50 concentration. The secondary antibody, biotin-conjugated donkey anti-rabbit (711-065-152), was purchased from Jackson ImmunoResearch (West Grove, PA, USA). Images of the antibody stained sections were taken using a Zeiss Axioskop MOT light microscope. Image processing was performed in Adobe Photoshop CS5.
5
Comprehensive Antibody Validation Techniques
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Western blotting analysis (WB): anti-HA (1:500, sc-805), anti-peripherin (1:500, sc-28539) and anti-vinculin (1:10000, sc-25336) were from Santa Cruz Biotechnology, Santa Cruz, CA, USA; anti-actin (1:5000, ab-8224) and anti-tubulin (1:6000, clone B512), were from Sigma-Aldrich, St-Louis, MO, USA, while anti-EGFR (1:2000, 20-ES04) was from Fitzgerald, Concord, MA, USA and anti-RILP (1:400, 13574–1-AP) from Proteintech, Rosemont, IL, USA. Immunofluorescence analysis: anti-early endosome antigen 1 (EEA1, 1:1000, ab70521, Abcam), anti-HA (1:500, ab9110, Abcam), anti-EGFR (1:100, 20-ES04, Fitzgerald). Secondary antibodies conjugated to fluorochromes for immunofluorescence or horseradish peroxidase (HRP) were from Invitrogen (Carlsbad, CA, USA), Santa Cruz Biotechnology or Fitzgerald. Immunohistochemistry: myelin basic protein (MBP, 1:100, ab7349, Abcam), anti-peripherin (1:1000, ab4666, Abcam), anti-vasoactive intestinal peptide (VIP, 1:400, ab22736, Abcam), anti-neurofilament 200 (NF-H, 1:400, N0142, Sigma-Aldrich), anti-PGP9.5 (1:500, MCA4750GA, Bio-Rad, Hercules, CA, USA), anti-EGFR (1:100, 20-ES04, Fitzgerald). Secondary antibodies Alexa Fluor-conjugated (Jackson ImmunoResearch, Cambridge, UK) were employed.
6
Multimodal Immunofluorescence Imaging of Neuronal Markers
7
Immunohistochemical Analysis of Fat Pads
8
Histological and Immunohistochemical Analyses
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Bones were fixed in 10% neutral buffered formalin for 48 h, decalcified in 10% EDTA for two weeks and stained with hematoxylin and eosin (HE). Lungs were fixed with Bouin’s solution and the number of metastatic foci was macroscopically counted.
For IHC, bones were fixed, decalcified, embedded in OCT compound and sectioned at 10μm thickness using a cryostat. After blocking, sections were incubated with the primary antibodies overnight at 4°C, and secondary antibodies for 60 min, mounted with coverslips in VECTASHIELD anti-fade mounting medium (Vector Laboraories Inc, Burlingame, CA) and observed under TCS SP8 confocal laser scanning microscope (Leica Microsystems, Nussloch, Germany)
Tumors and DRGs were sectioned at 10μm thickness, and incubated with primary antibodies to CD-31 (1:50, Abcam, #ab28364), DeadEnd™ Colorimetric TUNEL System (Promega, #G7360, Madison, WI), Ki67 (1:400, Cell Signaling, #9129), PGP9.5 (1:200, Abcam, #ab8189), CGRP (1:200 Abcam, #ab36001), TRPV1 (1:1,000 Abcam, #ab31895), peripherin (1:1,000 Abcam, #ab4666), or HGF (1:200, Abcam, #ab83760) overnight at 4°C, and a secondly fluorescent-labeled antibody (1:100) for 60 min or a streptavidin-biotin complex, EnVision HRP (Dako, Carpinteria, CA), for 60 min and visualized using a 3,3-diaminobenzidine (DAB) substrate-chromogen solution (Dako Cytomation Liquid DAB Substrate Chromogen System).
For IHC, bones were fixed, decalcified, embedded in OCT compound and sectioned at 10μm thickness using a cryostat. After blocking, sections were incubated with the primary antibodies overnight at 4°C, and secondary antibodies for 60 min, mounted with coverslips in VECTASHIELD anti-fade mounting medium (Vector Laboraories Inc, Burlingame, CA) and observed under TCS SP8 confocal laser scanning microscope (Leica Microsystems, Nussloch, Germany)
Tumors and DRGs were sectioned at 10μm thickness, and incubated with primary antibodies to CD-31 (1:50, Abcam, #ab28364), DeadEnd™ Colorimetric TUNEL System (Promega, #G7360, Madison, WI), Ki67 (1:400, Cell Signaling, #9129), PGP9.5 (1:200, Abcam, #ab8189), CGRP (1:200 Abcam, #ab36001), TRPV1 (1:1,000 Abcam, #ab31895), peripherin (1:1,000 Abcam, #ab4666), or HGF (1:200, Abcam, #ab83760) overnight at 4°C, and a secondly fluorescent-labeled antibody (1:100) for 60 min or a streptavidin-biotin complex, EnVision HRP (Dako, Carpinteria, CA), for 60 min and visualized using a 3,3-diaminobenzidine (DAB) substrate-chromogen solution (Dako Cytomation Liquid DAB Substrate Chromogen System).
9
Quantification of Peripherin Protein
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BMMSCs were washed and lysed, and the protein concentrations were measured using the BCA Protein Assay Kit (Thermo Scientific) with bovine serum albumin as the standard. Lysates (20 μg/lane) were separated by 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE); then, proteins were transferred to 0.22 μm hybridization nitrocellulose filter membranes (Merck Millipore Ltd.). The membranes were blocked in TBST containing 5% nonfat milk (w/v) for 3 h at room temperature. Peripherin was detected by western blot using a peripherin primary antibody (rabbit anti-peripherin antibody (ab4666), Abcam). After washing with TBST, the membrane was incubated with the corresponding secondary antibody (goat anti-rabbit IgG H&L (HRP) (ab205718), Abcam). The immunoreactive bands were detected using SuperSignal West Pico Chemiluminescent Substrate (Thermo Scientific), recorded on X-ray films (CWBIO), and then analyzed using ImageJ software (NIH, Bethesda, MD, USA). The GAPDH antibody (Cell Signaling Technology #5174) was used to monitor variation in loading of samples.
10
Antibody-Based Protein Detection Protocol
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Anti-RAGE antibody (anti-mouse, monoclonal #sc-80652) was purchased from SantaCruz Biotechnology, (Dallas, TX) anti-TLR4 (anti-mouse, monoclonal #ab22048 anti-HMGB1 antibody (anti-rabbit, monoclonal, ab79823), anti-CGRP antibody (anti-goat, polyclonal, #ab36001) and anti-peripherin antibody (anti-rabbit, #ab4666) were purchased from Abcam (Cambridge, MA). Anti-HMGB1 neutralizing antibody (Chicken IgY, # 326052233) and control antibody (Chicken IgY #326058471) were purchased from Shino test corporation (Tokyo, Japan). Anti-phospho-p44/42 MAPK antibody (anti-rabbit, monoclonal, #4370), anti-p44/42 MAPK antibody (anti-rabbit, monoclonal, #4695), anti-phospho CREB (anti-rabbit, monoclonal, #9198), horseradish peroxidase (HRP)-conjugated IgG antibody (anti-rabbit, monoclonal, #7074), HRP-conjugated IgG antibody (anti-mouse, monoclonal, #7076), anti-mouse IgG (H + L), F(ab′)2 Fragment (Alexa Fluor® 647 Conjugate) #4410, and anti-rabbit IgG (H + L), F(ab')2 Fragment (Alexa Fluor® 488 Conjugate) #4412 were purchased from Cell Signaling Technology (Danvers, MA).
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