At 28 days following the operation, the sciatic nerve tissues containing the area of crush injury site (5 mm away from the sciatic notch) were harvested and fixed with 4% paraformaldehyde. Then the longitudinal sections and transverse sections of the nerve tissue were prepared. Moreover, the sections were stained with NF200 (CST, 1:200), S100β (Abcam, 1:200), TuJ1 (Abcam, 1:200), MBP (Abcam, 1:200), CD31 (Abcam, 1:200), CD34 (Abcam, 1:200), VEGFR (Abcam, 1:200), GAPDH (Abcam, 1:200), Akt (Abcam, 1:500), p-AKT (Abcam, 1:500), PI3K (Abcam, 1:500), p-PI3K (ThermoFisher, 1:500) and PTEN (Abcam, 1:500). Secondary antibodies were as follows:Alexa Fluor568–conjugated Goat Anti-Rabbit IgG (Abcam), CoraLite594-conjugated Goat Anti-Mouse IgG (Proteintech, China), CoraLite488-conjugated Goat Anti-Rabbit IgG (Proteintech), CY3-labeled goat anti-rabbit (Servicebio) and AlexaFluor594-labeled goat anti-rabbit IgG (Abcam). Besides, we also used FITC-Tyramide (Servicebio) and CY3-Tyramide (Servicebio) to amplify fluorescence intensity. Nuclei were stained with DAPI, and the sections were observed under confocal laser scanning microscopy. The percentages of the markers positive areas were calculated by dividing integrated option density by selected region area, then multiplied by 100%. All parameters were measured using ImageJ.
Coralite594 conjugated goat anti mouse igg
Manufactured by Proteintech
Sourced in China, United States
CoraLite594-conjugated goat anti-mouse IgG is a secondary antibody that binds to mouse immunoglobulin G (IgG) and is conjugated with the CoraLite594 fluorescent dye. It is designed for use in immunofluorescence and other antibody-based detection assays.
Automatically generated - may contain errors
Lab products found in correlation
Coralite488 conjugated goat anti rabbit igg, by Proteintech (3 mentions)
P akt, by Abcam (1 mentions)
Vegfr, by Abcam (1 mentions)
P pi3k, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 568 conjugated goat anti rabbit igg, by Abcam (1 mentions)
Cy3 labeled goat anti rabbit, by Wuhan Servicebio Technology (1 mentions)
Fitc tyramide, by Wuhan Servicebio Technology (1 mentions)
Cy3 tyramide, by Wuhan Servicebio Technology (1 mentions)
Gapdh, by Abcam (1 mentions)
Ab64693, by Abcam (1 mentions)
Nbp2 25208, by Novus Biologicals (1 mentions)
Fluorescence microscope, by Olympus (1 mentions)
Edta na2, by Solarbio (1 mentions)
Anti phgdh, by Proteintech (1 mentions)
Anti mettl3, by Proteintech (1 mentions)
Anti hnrnpa2b1, by Proteintech (1 mentions)
Anti cdk6, by Abcam (1 mentions)
Anti slc1a4, by Proteintech (1 mentions)
Nutlin 3, by Merck Group (1 mentions)
Anti ki67, by Abcam (1 mentions)
6 protocols using coralite594 conjugated goat anti mouse igg
1
Nerve Injury Evaluation Using Immunostaining
2
Macrophage Phenotype Switching Identification
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All specimens were decalcified in 10% disodium Ethylenediamine tetraacetic acid (EDTA‐Na2, Solarbio, Beijing, China) at room temperature for ≈30 days. Subsequently, the decalcified samples were longitudinally embedded in paraffin wax and cut into 5 µm sections.
In order to detect phenotype switching of macrophages in vivo, immunofluorescence staining was performed. Tissue sections of 4 and 7 days were incubated with primary antibodies of anti‐CD86 (2 µg mL−1, NBP2‐25208, Novus) and anti‐CD206 (10 µg mL−1, ab64693, Abcam) at 4 °C overnight followed by incubation with the corresponding fluorescently labeled secondary antibody (CD86: CoraLite594‐conjugated Goat Anti‐Mouse IgG, CD206: CoraLite488‐conjugated Goat Anti‐Rabbit IgG; 1:200; Proteintech) and DAPI. The fluorescent images were obtained with a fluorescence microscope (Olympus), Image J was used to quantify the fluorescence intensity of the stained markers and normalized to DAPI‐stained nuclei counts.
In order to detect phenotype switching of macrophages in vivo, immunofluorescence staining was performed. Tissue sections of 4 and 7 days were incubated with primary antibodies of anti‐CD86 (2 µg mL−1, NBP2‐25208, Novus) and anti‐CD206 (10 µg mL−1, ab64693, Abcam) at 4 °C overnight followed by incubation with the corresponding fluorescently labeled secondary antibody (CD86: CoraLite594‐conjugated Goat Anti‐Mouse IgG, CD206: CoraLite488‐conjugated Goat Anti‐Rabbit IgG; 1:200; Proteintech) and DAPI. The fluorescent images were obtained with a fluorescence microscope (Olympus), Image J was used to quantify the fluorescence intensity of the stained markers and normalized to DAPI‐stained nuclei counts.
3
Comprehensive Protein Expression Analysis
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The following antibodies were used: anti-CDK1 (abcam, 2546 ab133327), anti-cyclin D1 (CST, 2922), anti-CDK6 (Abcam, ab151247), anti-cyclin E2 (CST, 4132), anti-PHGDH (Proteintech, 14,719–1-AP), anti-PSPH (Proteintech, 14,513–1-AP), anti-Ki-67 (Abcam, ab15580), anti-SLC1A4 (Proteintech, 13,067–2-AP), anti-p53(CST, 9282), anti-MDM2 (CST, 86,934), anti-hnRNPA2B1 (Proteintech, 14,813–1-AP), anti-Mettl3 (Proteintech, 15,073–1-AP), anti-GAPDH (CST, 2118), anti-Flag (CST, 2368), HRP goat anti-rabbit IgG (Biodragon, BF03008), HRP goat anti-mouse IgG (Biodragon, BF03001), CoraLite488-conjugated goat anti-rabbit IgG (Proteintech, SA00013-2), CoraLite594-conjugated goat anti-mouse IgG (Proteintech, SA00013-3), Nutlin-3 was purchased from Sigma (Shanghai, China).
4
Immunophenotyping of Human Decidua
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The human decidua was fixed in formalin immediately, followed by standard paraffin embedding. Immunofluorescence staining was performed overnight with the designated primary antibodies: anti-CD68 antibody (Abcam, ab31630), anti-CD206 antibody (Abcam, ab125028), anti-CK7 antibody (Servicebio, GB12225), anti-LDHA antibody (Abclonal, A1106), anti-CD86 antibody (Abclonal, A1199), anti-MCT-4 antibody (ProteinTech, 22787-I-AP) for 12 h at 4 °C. Secondary fluorescent antibodies: CoraLite594-conjugated goat anti-mouse IgG (ProteinTech, SA00013-3), fluorescein (FITC)-conjugated Affinipure goat anti-rabbit IgG (ProteinTech, SA00003-2), were added for 2 h, and DAPI (Servicebio, G1012, 1: 5000) was used for nuclear counterstaining. Samples were imaged through an immunofluorescence microscope (Olympus, Japan) 24 h after mounting.
5
Immunofluorescence Staining of α-SMA
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The CFSC cells were fixed with 4% PFA and stained with the mouse anti-α-SMA antibody (Sigma Aldrich, Shanghai, China), then stained with the CoraLite594-conjugated goat anti-mouse IgG (Proteintech, Wuhan, China). The nuclei were counterstained with DAPI (Solarbio, Beijing, China). A Zeiss Axio Observer 5 microscope (Carl Zeiss, Oberkochen, Germany) was used for the observations.
6
Immunocytochemical and Immunofluorescent Staining of NIS and PTEN
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Immunocytochemical staining/ICC was performed on the cell-bearing coverslips obtained from each of the experimental groups by the method described elsewhere 31 (link). The antibodies used were: NIS (Bioss, bs-0448R, Beijing, China; 1:500), PTEN (Wanleibio, WL01901, Shenyang, China; 1:250), E-cadherin (Proteintech, 20874-1-AP, Chicago, USA; 1:500). The color reaction was performed by using 3,30-diaminobenzidine tetrahydrochloride (DAB). For double immunofluorescent staining/IF, after washed with PBS 3 times and then blocked by normal goat serum for 30 minutes at 37°C, the cell-bearing coverslips were co-incubated with rabbit anti-NIS (1:250) and mouse anti-PTEN (Bioss, bsm-33320M, Beijing, China; 1:250) overnight at 4°C in a humid chamber. Then, the coverslips were co-incubated with coralite488-conjugated affinipure goat anti-rabbit IgG (1:500, Proteintech, SA00013-2, Chicago, USA) and coralite594-conjugated goat anti-mouse IgG (1:500, Proteintech, SA00013-3, Chicago, USA) at 37°C for 60 minutes in the dark. Cell nucleus were stained with Hoechst, sealed with fluorescence mounting medium, and observed and imaged under a positive fluorescence microscope (Zeiss, Ax10 Axio, Germany).
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