Mice were sacrificed at various time points (days 3, 5, and 7) of the experiments, and skin tissues were harvested so that part of the tissue was fixed in formalin-PBS buffer, paraffin-embedded, and sectioned to generate wound-edge specimens of 4 µm diameter. After de-paraffinized, sections were stained with Masson’s trichrome by standard procedures and examined under light microscopy. For immunofluorescence staining, antigen retrieval was performed using citrate buffer, pH to 6.0, and microwaving for 5 min then cooling for 3 min. After non-specific blocking, specific staining was performed by using α-SMA (Sigma-Aldrich, St. Louis, MO, USA), Pro-collagen 1A1 (Santa Cruz, CA) Ab, or SM22α (Abcam, USA) Ab followed by incubation with secondary antibody alexa fluor 594-conjugated IgG or Texas red (Invitrogen, Molecular Probe, Carlsbad, CA). Counterstaining was performed with DAPI (Invitrogen) and imaged by using a fluorescent microscope (Nikon E800 with MetaMorph version 4.5 software, Universal Imaging Corp.). The GFP staining procedure was carried out according to the protocol of VECTASTAIN Elite ABC kit (Vector laboratories Inc, Burlingame, CA) after using anti- GFP primary antibody (Zymed, Invitrogen). Immunohistochemical images were analyzed using an image analysis software program (ImageJ, NIH).
Anti gfp primary antibody
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Anti-GFP primary antibody is a laboratory reagent used to detect and quantify the presence of green fluorescent protein (GFP) in samples. It is a highly specific antibody that binds to the GFP protein, allowing for its identification and visualization in various experimental applications.
Automatically generated - may contain errors
Lab products found in correlation
Anti ph3 primary antibody, by Santa Cruz Biotechnology (2 mentions)
Alexa fluor 555 conjugated anti rabbit and alexa fluor 488 conjugated anti mouse secondary antibodies, by Thermo Fisher Scientific (2 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (2 mentions)
Sm22α, by Abcam (1 mentions)
Texas red, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 594 conjugated igg, by Thermo Fisher Scientific (1 mentions)
α sma, by Merck Group (1 mentions)
Vectastain elite abc kit, by Vector Laboratories (1 mentions)
Metamorph, by Universal Imaging (1 mentions)
Rat anti brdu antibody, by Abcam (1 mentions)
Bromodeoxyuridine (brdu), by Merck Group (1 mentions)
Lsrfortessa analyzer, by BD (1 mentions)
40 μm mesh cell strainer, by BD (1 mentions)
Dithiothreitol (dtt), by Thermo Fisher Scientific (1 mentions)
Liberase, by Roche (1 mentions)
Alexa fluor 488 secondary antibody, by Thermo Fisher Scientific (1 mentions)
Bz x700, by Keyence (1 mentions)
Alexa fluor 488 conjugated secondary antibody, by Jackson ImmunoResearch (1 mentions)
Cytoseal, by Thermo Fisher Scientific (1 mentions)
9 protocols using anti gfp primary antibody
1
Skin Wound Healing Histological Analysis
2
Whole-mount Immunohistochemistry Protocol
3
GFP Immunohistochemistry in Brain
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Animals were perfused with 4% paraformaldehyde at 4 or 8 weeks post-injection. The brains were then removed, postfixed, cryoprotected in 30% sucrose, and frozen. Coronal sections (50 µm thick) were cut and fluorescence immunohistochemistry was performed on free-floating sections. For immunodetection of GFP, polyclonal rabbit anti-GFP primary antibody (Invitrogen, 1:1000) and secondary antibody Alexa Fluor 488- (GFP experiment) or 568-conjugated (PSD95 experiment) goat anti-rabbit (1:1000) were used. To counterstain nuclei, the tissue was incubated in DAPI.
4
BrdU Incorporation Assay in Zebrafish Embryos
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Embryos at either 48- or 72 hpf were incubated in 10mM BrdU (Sigma Aldrich) for 20 min on ice, washed in egg water and incubated at 28°C for 3 h (48 hpf) or 5h (72 hpf). The embryos were then used for immunohistochemistry with anti-GFP primary antibody (Invitrogen, 1:200) and anti-rabbit secondary antibody conjugated to Alexa Fluor 488 (Invitrogen, 1:200), followed by fixation with 4% paraformaldehyde at room temperature for 15 min. The embryos were then cut and their heads were used for genotyping. Embryos of the same genotype were pooled together and subjected to washes with water followed by treatment with 2N HCl for 1h at room temperature. The embryos were then washed consecutively with PBS-Tween and 1M Tris-HCl pH 9.5 and subjected to immunohistochemistry against BrdU with rat anti-BrdU antibody (Abcam, 1:200) and anti-rat antibody conjugated to Cy3 (Millipore, 1:400). Fluorescence was visualized using confocal microscopy.
5
Whole-mount Immunohistochemistry Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Whole-mount immunohistochemistry was performed using standard methods (Li et al., 2012 (link)). Anti-pH3 primary antibody (Santa Cruz) was used at 1:3000 dilution. Anti-GFP primary antibody (Invitrogen) was used at 1:1000 dilution. Alexa Fluor 555-conjugated anti-rabbit and Alexa Fluor 488-conjugated anti-mouse secondary antibodies (Invitrogen) were used at 1:2000 dilution.
6
Flow Cytometry of Zebrafish CHT Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The progeny from a Tg(c-myb:EGFP/nol9+/sa1022 x nol9+/sa1022 cross at 96 hpf was screened for the presence of EGFP and subjected to immunohistochemical staining with anti-GFP primary antibody (Invitrogen, 1:200) and anti-rabbit secondary antibody conjugated with Alexa Fluor 488 (Invitrogen, 1:200). Afterwards, each larva was dissected: the CHT was stored and the rest of the body was used for genotyping for the nol9sa1022 allele. CHTs from 8 larvae of the same genotype were pooled, incubated for 30 min with 10 mM DTT (Life Technologies) in Danieau’s solution, followed by incubation with 50 μg/ml liberase (Roche) in PBS for 3 h at 37°C. Single-cell suspension was prepared by passing the solution through a 40 μm mesh cell strainer (Becton Dickinson). Flow cytometry was performed on a BD LSR-Fortessa analyzer (Becton Dickinson). Non-transgenic siblings subjected to immunohistochemical staining were used as a negative control.
7
GFP Immunostaining of VTA Slices
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
GFP immunofluorescence was performed to enhance the GFP expression following stereotaxic injection of AAV-GFP and AAV-Cre-GFP. VTA slices (40 µm) were blocked with 10% normal goat serum (NGS) and incubated with a polyclonal anti-GFP primary antibody (rabbit, 1:1000, Invitrogen, Waltham, MA, USA) overnight at 4 °C and incubated with a polyclonal AlexaFluor 488 secondary antibody (rabbit, 1:200, Invitrogen, Waltham, MA, USA) for 2 h at room temperature. GFP immunostaining was imaged on a Keyence BZ-X700 (Keyence, Itasca, IL, USA) and analyzed using the Fiji distribution of ImageJ (NIH). Images were converted to binary masks prior to analysis and Integrated Density values were used to define signal intensity within a region of interest. Binary masks cause inversion of pixel values, thus greater ID indicates lower GFP signal.
8
Immunostaining of GFP-expressing cells
9
Immunohistochemical Staining of GFP Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Sections (three per each 6-mm block, with a separation of 2 mm) were washed three times in PBST for 5 minutes each time and then treated with 1% H2O2 for 20 minutes. Sections were incubated in Sniper blocking solution (http://biocare.net/product/background-sniper/ ) for 30 minutes at room temperature and then incubated overnight with the primary anti-GFP antibody (https://www.lifetechnologies.com/ ) diluted 1:1,000 in Da Vinci Green Diluent (http://biocare.net/ ). After three rinses in PBS containing 0.1% Tween-20 (PBST) for 5 minutes each time, sections were incubated in Mach 2 horseradish peroxidase (HRP) polymer (http://biocare.net/ ) for 1 hour and then washed three times before colorimetric development with 3,3’-diaminobenzidine. Immunostained sections were counterstained with cresyl violet, and mounted on slides and sealed with Cytoseal (http://www.thermoscientific.com/ ).
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!