The prepared slides were rehydrated, and immumohistochemistry staining (IHC) was applied to examine the protein expressed level of Collagen I (COL-I), Osteocalcin (OCN) and Bone morphogenetic protein-2 (BMP-2). Briefly, the rehydrated slides were heated in sodium citrate buffer (Abcam) for antigen retrieval. Then the slides were washed with PBS, followed by being exposed to 5% BSA for 1 h at 37 °C to block non-specific binding. After that the slides were incubated with primary antibodies at 4 °C overnight, followed by incubation with 50 μL of mouse-anti rabbit-horseradish peroxidase (HRP) (BOSTER) for 50 min at room temperature. The reaction products were visualized with 3-3′ diaminobenzidine (DAB) (BOSTER) according to the manufacturer's instructions. The antibodies were diluted as follows: OCN antibody (1: 500, Abcam), BPM-2 antibody (1: 200, Beyotime) and COL-І antibody (1: 200, Beyotime). Quantification data of IHC were performed by calculating the mean optical density value of positive-staining areas. The area of interest was firstly focused on the new formed issue between the host bone and cement implant, then areas (n = 5) with remarkably high immunohistochemical staining were selected and manually quantified using the Image-Pro Plus software (Media Cybernetics).
Sodium citrate buffer
Manufactured by Abcam
Sodium citrate buffer is a commonly used buffer solution that maintains a stable pH environment. It is primarily used to control the acidity or basicity of a solution in various laboratory applications.
Automatically generated - may contain errors
Lab products found in correlation
Ocn antibody, by Abcam (1 mentions)
3 3 diaminobenzidine dab, by Boster Bio (1 mentions)
Image pro plus software, by Media Cybernetics (1 mentions)
Anti vwf antibody, by Merck Group (1 mentions)
Normal goat serum (ngs), by Vector Laboratories (1 mentions)
Mouse anti rat cd68 antibody, by Bio-Rad (1 mentions)
3 protocols using sodium citrate buffer
1
Evaluating Bone Implant Integration
2
Femoral Artery Histological Analysis
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Femoral arteries were perfusion fixed using paraformaldehyde, and then carefully excised along with a block of the surrounding muscle to save neighbor vasculature in the connective tissues. Excised tissues were then carefully fixed in 4% paraformaldehyde, embedded in the paraffin-block and serially sectioned. Cross-sections of the paraffin block were stained with hematoxylin and eosin (H&E) and Von Willebrand Factor (vWF) for endothelium staining. In briefly, for antigen retrieval, treatment dewaxed slides were heated in sodium citrate buffer (pH 6.0, abcam, Cambridge, MA) at 95 °C for 30 min in blocking solution of goat serum and incubated with the anti-vWF antibody (Millipore, Burlington, MA) at 4 °C overnight. Incubated slides were washed with PBS 3 times for 3 mins. The slides were incubated with Cy3-conjugated IgG (Jackson ImmunoResearch Laboratories, West Grove, PA) and counterstained with DAPI. The stained slides were observed by using a fluorescent microscope (IX-81, Olympus, Center Valley, PA). Brightness (+40%) and contrast (+40%) of acquired fluorescence images were adjusted to improve visibility by using ImageJ software43 (link).
3
Immunohistochemical Analysis of Tumor-Associated Macrophages
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Tumor sections were deparaffinized in xylene and rehydrated, followed by antigen retrieval with retrieval buffer (sodium citrate buffer, pH 6.0; Abcam). The peroxidase activity was inhibited by 3% hydrogen peroxide for 10 min and the sections were incubated with 10% normal goat serum (Vector Laboratories, Burlingame, CA, USA) to block the non-specific binding of reagents. Rat anti-mouse CD68 antibody (1:100, Bio-Rad, Hercules, CA, USA) was applied as primary antibody overnight in a moist chamber at 4°C. Goat anti-rat immunoglobulin (1:100, Abcam) was applied as secondary antibody for 2 h at 37°C, followed by streptavidin-HRP for detection. Immunostaining was developed with DAB + NI-Chromogen substrate (Vector Laboratories), followed by counterstaining with methyl green. Tumor tissue was examined under the light microscope at magnification 40X and 100X.
CD68-positive cells were developed for light microscopy with DAB + NI-Chromogen substrate (dark-brown staining). Other cells stained green with methyl green (Figures 2 3
CD68-positive cells were developed for light microscopy with DAB + NI-Chromogen substrate (dark-brown staining). Other cells stained green with methyl green (
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