Dissociated DRG neurons cultured on coverslips were rinsed once with 1x Recording buffer (25 mM HEPES pH7.4, 120 mM NaCl, 5 mM KCl, 2 mM CaCl2, 1 mM MgCl2, 30 mM Glucose) and fixed in 4% paraformaldehyde (PFA)/sucrose for 10 min at room temperature. Samples were permeabilized in PBS containing 0.25% (w/v) Triton-X-100 for 10 min at 4 °C, blocked with PBS containing 10% (v/v) Normal Goat Serum (Southern Biotech, 0060-01) for 1 h and incubated overnight at 4 °C with primary antibodies diluted in blocking solution. After three washes with PBS, cells were incubated for 1 h at room temperature with AlexaDye-conjugated fluorescent secondary antibodies diluted in blocking solution, prior to three final PBS washes and mounting in FluorSave reagent (Millipore Sigma).
Normal goat serum (ngs)
Manufactured by Southern Biotech
Sourced in United States
Normal goat serum is a biological product derived from the blood of healthy goats. It is a complex mixture of proteins, antibodies, and other substances that can be used in various laboratory applications as a supplement or control.
Automatically generated - may contain errors
Lab products found in correlation
Nitrocellulose blotting membrane, by Cytiva (2 mentions)
Ripa buffer, by Roche (2 mentions)
Na3vo4, by Merck Group (2 mentions)
Ripa buffer, by Boston BioProducts (2 mentions)
Protease inhibitor cocktail, by Roche (2 mentions)
Enhanced chemi luminescence (ecl), by PerkinElmer (2 mentions)
Clone x53, by Thermo Fisher Scientific (2 mentions)
Triton x 100, by Thermo Fisher Scientific (2 mentions)
Mini protean tgx gel, by Bio-Rad (2 mentions)
Fluorsave reagent, by Merck Group (1 mentions)
Sb225002, by Bio-Techne (1 mentions)
Mouse igg1 anti ctbp2, by BD (1 mentions)
Alexa fluor 488 goat anti mouse igg2a, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 647 donkey anti goat igg, by Thermo Fisher Scientific (1 mentions)
Normal goat serum (ngs), by Agilent Technologies (1 mentions)
Dmra microscope, by Leica (1 mentions)
Prolong diamond antifade mountant with dapi, by Thermo Fisher Scientific (1 mentions)
Paraformaldehyde (pfa), by Electron Microscopy Sciences (1 mentions)
Nis elements acquisition software, by Nikon (1 mentions)
Cytation 5 cell imaging multi mode reader, by Agilent Technologies (1 mentions)
12 protocols using normal goat serum (ngs)
1
Immunocytochemistry of Dissociated DRG Neurons
2
Quantifying Tumor Cell Proliferation
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To evaluate the proliferative indices of xenograft tumors, we evaluated the nuclear protein Ki-67/MIB-1 that it is expressed in all active phases of the cell cycle but is absent in quiescent cells. Slides were treated post-fixation with ice-cold 70% methanol, blocked for 1 hour in 5% normal goat serum (SouthernBiotech, Birmingham, AL) and hybridized with a mouse monoclonal anti-MIB1 antibody (Dako, Agilent Technologies, Carpinteria, CA) for 1 hour at RT, followed by goat anti-mouse Alexa Fluor 488 fluorescent antibody (Invitrogen). Pictures were taken with a Leica EL6000 attached to a Leica DMI3000B inverted microscope (Leica Mycrosystems).
3
Comprehensive Antibody Sourcing Protocol
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SB225002 was purchased from TOCRIS bioscience (#2725/10). Antibodies used in this study were purchased from different companies: anti-CtBP2 mouse IgG1 was purchased from BD Biosciences (#612044), Rabbit polyclonal Myosin-VIIa was obtained from Proteus Biosciences (#25-6790), Rabbit polyclonal IgG CXCL1 (ab269939), Rabbit monoclonal IBA1 (ab178846) and Mouse monoclonal IgG1 CD68 (ab31630) were purchased from Abcam, Mouse monoclonal IgG2a CD45 (05-1410) and anti-glutamate receptor 2 (GluR2) IgG2a were purchased from Millipore (#MAB397). Rhodamine (TRITC) AffiniPure Donkey Anti-Rabbit IgG secondary antibody was purchased from Jackson ImmunoResearch Laboratories (711-025-152). Secondary antibodies used were as follows: Alexa FluorTM 568 goat anti-mouse IgG1 (A-21124), Alexa FluorTM 488 goat anti-mouse IgG2a (A-21131) and Alexa FluorTM 647 donkey anti-goat IgG (A-21447) were purchased from Life Technologies. All primers were purchase from integrated DNA technologies (IDT DNA) (Iowa. USA). Normal Goat serum was purchased from SouthernBiotech (0060-01) (Alabama. USA).
4
Analyzing Phosphorylated PD-1 in Jurkat and EL4 Cells
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Jurkat hPD-1 or EL4 (10 × 106 cells) were treated for 5 minutes at 37°C with 0.1 mmol/L pervanadate (premixing orthovanadate, Na3VO4 (Sigma Aldrich) and H2O2 (Sigma Aldrich) for 15 minutes at room temperature to generate pervanadate), and cells were lysed in RIPA buffer (Boston Bioproducts) supplemented with Protease Inhibitor Cocktail (Roche; one tablet in 10 mL of RIPA buffer) and lysates were prepared (36–39 (link)). Cell lysates (20 μg) were separated on 4% to 15% Mini-Protean TGX gels (Bio-Rad) and transferred onto nitrocellulose blotting membranes (Amersham). A blocking buffer of 5% milk (Fisher) in 1XTBS-T (1X TBS + 0.1% Tween-20) was used to block nonspecific binding by incubation for 1 hour at room temperature. Primary antibodies against phospho–PD-1 (clone 407.6G12), anti-mouse PD-1 (clone 29F.1A12; ref. 35 (link)), or anti-human PD-1 (clone EH33; ref. 40 (link)) diluted to 2 μg/mL in 1% BSA in TBS-T were incubated with the membrane overnight at 4°C. After washing three times with TBS-T, the membrane was incubated with HRP-conjugated goat anti-mouse IgG or anti-rat IgG secondary antibody (Southern Biotech) diluted 1:5000 in 2.5% milk in TBS-T buffer plus 1% normal goat serum (Southern Biotech) for 1 hour at room temperature. Immunoreactive proteins were detected using enhanced chemiluminescence reagent ECL (PerkinElmer; ref. 41 (link)).
5
Immunofluorescence Analysis of Porcine Hepatocytes
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Porcine hepatocyte-like cells were fixed in 4% paraformaldehyde for 20 minutes, and then the cells were permeabilized with 0.1% Triton X-100 for 10 minutes at room temperature. Slides were blocked with 10% normal goat serum (SouthernBiotech) for 30 minutes. The cells were subsequently incubated overnight at 4°C with anti-human albumin (ALB) (Sigma-Aldrich) or anti-human alpha fetoprotein (AFP) (Dako) antibodies at 1∶200 dilution rate in 3% normal goat serum. This was followed by incubation with fluorescein secondary antibody (Invitrogen) for 2 hours at room temperature. Fluorescence images were visualized and captured using a Leica DMRA microscope (Leica).
6
GAG and Collagen Immunohistochemistry Protocol
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Fixed pellets were paraffin embedded and sectioned. Deposition of
glycosaminoglycan (GAG) was determined by thionine staining.23
For immunohistochemical stainings, sections were pretreated with 0.1% w/v
pronase and 1% w/v hyaluronidase and blocked using normal goat serum
(Southern Biotech, Birmingham, AL, USA). Sections were incubated with
either mouse monoclonal antibody against collagen type II 0.4 μg/mL
(Developmental Studies Hybridoma Bank, Cat. #II-II6B3) for 60 minutes
or collagen type X 5 μg/mL (ThermoFisher, Clone X53, Cat. #14-9771-82)
in PBS 1% BSA overnight. After incubation with a biotinylated goat
anti-mouse antibody and ALP-conjugated streptavidin, staining was
revealed by incubation with a New Fuchsin substrate (Chroma, Kongen,
Germany). As corresponding isotype controls 0.4 μg/mL and 5 μg/mL of
an isotype immunoglobulin G1 monoclonal antibody were used.
glycosaminoglycan (GAG) was determined by thionine staining.23
For immunohistochemical stainings, sections were pretreated with 0.1% w/v
pronase and 1% w/v hyaluronidase and blocked using normal goat serum
(Southern Biotech, Birmingham, AL, USA). Sections were incubated with
either mouse monoclonal antibody against collagen type II 0.4 μg/mL
(Developmental Studies Hybridoma Bank, Cat. #II-II6B3) for 60 minutes
or collagen type X 5 μg/mL (ThermoFisher, Clone X53, Cat. #14-9771-82)
in PBS 1% BSA overnight. After incubation with a biotinylated goat
anti-mouse antibody and ALP-conjugated streptavidin, staining was
revealed by incubation with a New Fuchsin substrate (Chroma, Kongen,
Germany). As corresponding isotype controls 0.4 μg/mL and 5 μg/mL of
an isotype immunoglobulin G1 monoclonal antibody were used.
7
Western Blot Analysis of Phospho-PD-1 in T Cells
8
Cytospins and Immunofluorescence of Neutrophils
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Cytospins (Shandon CytoSpin cytocentrifuge) were stained with May-Grünwald-Giemsa solution and analyzed using a Cytation 5 cell imaging multimode reader (BioTek) with a 40× objective.
For immunofluorescence microscopy, neutrophils were settled onto coverslips coated with poly-l -lysine for 1 h at 37°C. The cells were infected for 30 min or 3 h, and the cells were fixed with 4% PFA (Electron Microscopy Sciences). Neutrophils were permeabilized with 0.5% Triton X-100 (Fisher Scientific), blocked with 5% normal goat serum (Southern Biotech), and stained overnight with an anti-myeloperoxidase (anti-MPO) (N4C7[989B]; BioLegend) primary antibody. An Alexa Fluor 594 (AF594)-conjugated secondary antibody (Life Technologies) was used. Coverslips were mounted onto glass slides using ProLong Diamond antifade mountant with DAPI (Life Technologies). Images were acquired using a Nikon Eclipse Ti inverted microscope with a 60× objective and NIS-Elements acquisition software (Nikon Instruments).
For immunofluorescence microscopy, neutrophils were settled onto coverslips coated with poly-
9
Neutrophil Immunofluorescence Microscopy
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For immunofluorescence microscopy, neutrophils were settled onto coverslips coated with poly-l -lysine (Corning) for 30 min at 37°C. Infections were done for 16 h, and cells were fixed with 4% paraformaldehyde (PFA; Electron Microscopy Sciences). Neutrophils were permeabilized with 0.5% Triton X-100 (Fisher Scientific), blocked with 5% normal goat serum (Southern Biotech), and stained overnight with an anti-myeloperoxidase (MPO, N4C7[989B]; BioLegend) primary antibody, followed by an AF594-conjugated secondary antibody (Life Technologies). Coverslips were mounted onto glass slides using Vectashield with DAPI (4′,6-diamidino-2-phenylindole; Vector Laboratories). Images were acquired using a Leica SP8 confocal microscope with a 63× oil objective. Images were analyzed using FIJI (81 (link)).
10
Immunofluorescence Protocol for Stem Cells
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Cells were fixed with 4% paraformaldehyde (PFA) (Sigma) for 10 min; permeabilized with 0.1% Triton X/PBS for 1 hr at room temperature; blocked with 5% Normal Goat Serum (SouthernBiotech), 0.1% Tween 20 in PBS for 1 hr; and stained overnight with the primary antibody at 4°C (1:1,000 in blocking solution) and 1 hr with the secondary antibodies (1:1,000 in blocking solution). Anti-mouse NANOG and DPPA4 antibodies were obtained from eBioscience (14-5761) and Cosmo Bio (CAC-TMD-PB-DP4), respectively.
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