Recombinant PbGAPDH fragment or pET32b tag protein was attached to a Ni-coated ELISA plate (Thermo Fisher Scientific). After washing and blocking with 1% BSA in PBS, the coated wells were further incubated with lysate of 293T7 cells which were transfected to express rat CD68. An anti-S tag antibody determined the amount of PBGAPDH fragments or pET32b tag protein attached to the wells. The amount of CD68 binding to the attached protein was determined with an anti-rat CD68 antibody (ABD SEROTEC) and normalized to the amount of attached protein. For the pull-down assays, recombinant rat CD68 was bound to protein-A beads (Invitrogen) using anti-rat CD68 antibody (ABD SEROTEC). After incubation with recombinant PBGAPDH fragments or pET32b tag protein, the CD68-Protein A beads were boiled in Laemmli buffer to elute bound proteins. Western blotting assays with an anti-thioredoxin tag antibody visualized the recombinant PBGAPDH G3 and G6 fragments and pET32b tag proteins.
Anti rat cd68 antibody
Manufactured by Bio-Rad
Sourced in United Kingdom
The Anti-rat CD68 antibody is a monoclonal antibody that specifically binds to the CD68 antigen expressed on the surface of macrophages and monocytes in rats. CD68 is a glycoprotein that is commonly used as a marker for the identification and quantification of macrophages in various tissues and cell types.
Automatically generated - may contain errors
Lab products found in correlation
Protein a bead, by Thermo Fisher Scientific (3 mentions)
Ni coated elisa plate, by Thermo Fisher Scientific (3 mentions)
Sp8 laser scanning confocal microscope, by Leica (1 mentions)
Las x software, by Leica (1 mentions)
Vt1000s vibratome, by Leica (1 mentions)
Vectashield antifade mounting medium, by Vector Laboratories (1 mentions)
Goat anti rat secondary antibody conjugated to alexa fluor 594, by Thermo Fisher Scientific (1 mentions)
Rat anti f4 80 antibody, by Bio-Rad (1 mentions)
Alexa fluor 555 donkey anti rat igg, by Thermo Fisher Scientific (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (1 mentions)
Image pro plus 6, by Media Cybernetics (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions)
Tissue tek, by Sakura Finetek (1 mentions)
Horseradish peroxidase conjugated anti mouse igg, by Agilent Technologies (1 mentions)
Oct compound, by Sakura Finetek (1 mentions)
Coolscope, by Nikon (1 mentions)
8 protocols using anti rat cd68 antibody
1
Characterization of CD68-GAPDH Interaction
2
Characterization of PbGAPDH-CD68 Interaction
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Recombinant PbGAPDH fragment or pET32b tag protein was attached to a Ni-coated ELISA plate (Thermo Fisher Scientific). After washing and blocking with 1% BSA in PBS, the coated wells were further incubated with lysate of 293T7 cells which were transfected to express rat CD68. An anti-S tag antibody determined the amount of PbGAPDH fragments or pET32b tag protein attached to the wells. The amount of CD68 binding to the attached protein was determined with an anti-rat CD68 antibody (AbD Serotec) and normalized to the amount of attached protein. For the pull-down assays, recombinant rat CD68 was bound to protein-A beads (Invitrogen) using anti-rat CD68 antibody (AbD Serotec). After incubation with recombinant PbGAPDH fragments or pET32b tag protein, the CD68-Protein A beads were boiled in Laemmli buffer to elute bound proteins. Western blotting assays with an anti-thioredoxin tag antibody visualized the recombinant PbGAPDH G3 and G6 fragments and pET32b tag proteins.
3
Characterizing CD68 Protein Interactions
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PbGAPDH or pET32b tag protein was attached to a Ni-coated ELISA plate (Thermo Fisher Scientific). After washing, the coated wells were further incubated with the membrane protein fraction of Cos7 cells (a monkey kidney cell line), which were either transfected to express rat CD68 or not transfected as a negative control. An anti–thioredoxin tag antibody determined the amount of PbGAPDH or pET32b tag protein attached to the wells. The amount of CD68 binding to the attached protein was determined with an anti–rat CD68 antibody (AbD Serotec) and normalized to the amount of attached protein.
Recombinant rat CD68 was bound to protein A beads (Invitrogen) using anti–rat CD68 antibody (AbD Serotec). After incubation with either PbGAPDH or pET32b tag protein, the CD68–protein A beads were boiled in Laemmli buffer to elute bound proteins. Western blotting assays with an anti–thioredoxin tag antibody visualized the recombinant PbGAPDH and pET32b tag proteins.
Recombinant rat CD68 was bound to protein A beads (Invitrogen) using anti–rat CD68 antibody (AbD Serotec). After incubation with either PbGAPDH or pET32b tag protein, the CD68–protein A beads were boiled in Laemmli buffer to elute bound proteins. Western blotting assays with an anti–thioredoxin tag antibody visualized the recombinant PbGAPDH and pET32b tag proteins.
4
Confocal Imaging of Infected Lung Tissue
5
Immunofluorescence Liver Staining Protocol
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Immunofluorescence was used to detect F4/80 and CD68 as described previously 20 . Briefly, 8 µm sections were cut from frozen livers and fixed for 10 minutes in 4% formalin. The sections were then incubated in blocking buffer (10% goat serum; 1 hour) followed by incubation with either rat anti-F4/80 antibody (Bio-Rad) diluted 1:500 or rat anti-CD68 antibody (Bio-Rad) diluted 1:500 overnight at 4°C. After washing, the sections were incubated with goat anti-rat secondary antibody conjugated to Alexa Fluor 594 for 1 hour (diluted 1:500, Thermo Fisher Scientific). Proliferating cell nuclear antigen (PCNA) was detected as described previously 20 .
6
Immunofluorescence Staining of CD68 in Tissues
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The slides were rinsed with PBS and masked in a solution that contains 10% goat serum at ambient temperature, followed by an incubation period at 4°C with rat anti-CD68 antibody (1 : 250, Serotec, Oxford, UK). Finally, the sections were treated with Alexa Fluor 555-donkey anti-rat IgG for 45 minutes at 37°C (1 : 1000, Invitrogen, Carlsbad, USA). 4′,6-diamidino-2-phenylindole (DAPI; 1 : 500, Invitrogen) was used to counterstain the nuclei for 30 seconds. A confocal microscope was used to capture the fluorescent pictures (Leica SP5, Germany). The intensity of immunofluorescence staining was measured using the Image-Pro Plus 6.0 software (Media Cybernetics, USA) to calculate the integrated optical density (IOD) of positive expression from six randomly selected sections, as described previously [16 (link)].
7
Sciatic Nerve Injury and Immunostaining
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At the predetermined time points, mice were deeply anesthetized with isoflurane (3% in 95% O 2 ) and intracardially perfused with 4% paraformaldehyde in 0.1 M phosphate-buffered solution. Ipsilateral (injured and sham, respectively) and contralateral (control) sciatic nerves were harvested and immediately placed in phosphate-buffered saline buffered 4% paraformaldehyde (pH 7.4; Sigma-Aldrich, Darmstadt, Hesse, Germany) for 3 h at 4°C. Sciatic nerve samples were then embedded in Tissue Tek (Sakura Finetek, Staufen im Breisgau, Baden-Württemberg, Germany), snap-frozen in liquid nitrogen-cooled methyl butane (Sigma-Aldrich) and stored at -80°C. Ten-micrometer thick transversal sections were prepared from the cryoblock at the site of the crush injury and stored at -80°C until used. Fluorescence immunohistochemistry on frozen sections was performed for 16-18 h at 4°C using the rat anti-CD68 antibody (Serotec, Puchheim, Bavaria, Germany) (dilution 1:250), followed by incubation for 45 min with the secondary species-specific antibody (dilution 1:250) conjugated with Alexa Fluor 594. Sections were mounted and counterstained with 4′,6′-diamidino-2-phenylindole (DAPI) (Sigma-Aldrich).
8
Evaluation of Scaffold Encapsulation in Rat
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Scaffolds implanted in rat subcutaneous tissue were resected with surrounding tissue, embedded in OCT compound (Sakura Finetek, Tokyo, Japan), and sectioned by cryostat. Hematoxylin and eosin staining and immunostaining for CD68, which was present on the macrophage, were performed. For immunostaining, anti-rat CD68 antibody (AbD Serotec, Oxford, UK) as the first antibody and horse radish peroxidase-conjugated anti-mouse IgG as the secondary antibody (DAKO, Glostrup, Denmark) were used. Stained samples were observed and photographed by Coolscope (Nikon, Tokyo, Japan). Encapsulation thickness around implanted scaffold was measured on picture of HE staining. In brief, three points of fibrous connective tissue existing as thin membrane between scaffold and host body wall were measured on picture. Average encapsulation thickness was calculated and each value was statistically compared with the thickness of non-coat scaffold by student’s t test.
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