Tissue sections were deparaffinized in xylene, and hydrated in graded ethanol solutions. Endogenous peroxidase activity was blocked by incubating the sections in 3% hydrogen peroxide in PBS for 10 min. Nonspecific binding was blocked in 5% bovine serum albumin (BSA) in PBS for 60 min. Then, the sections were incubated in rabbit anti-laminin (1:1,200, Sigma-Aldrich), rabbit anti-PCNA (1:1,000; Cell Signaling, Danvers, MA, USA) or biotinylated DBA lectin (1:300; Sigma-Aldrich) overnight at 4°C. After washing in PBS 3 times for 5 min each time, the sections were incubated with a secondary antibody for 60 min at 37°C followed by fresh diaminobenzidine (ZSGB-BIO, Beijing, People’s Republic of China) solution, together with counter-staining with Harris’ hematoxylin. Experimental results were observed by light microscopy, and analyses of immunohistochemical staining were taken using an NIS-Elements analysis system (Nikon). Each slide was measured in a randomly selected 10 fields under the same magnification (×400) and light intensity. Mean values of optical density for positive cells were calculated using 5 placenta sections from 10 individuals in each group.
Biotinylated dba lectin
Manufactured by Merck Group
Sourced in United States
Biotinylated DBA lectin is a laboratory reagent used for the detection and analysis of glycoprotein structures containing N-acetylgalactosamine. It is a binding protein derived from the seeds of the Dolichos biflorus plant. The biotinylation of the lectin allows for its detection and labeling in various experimental techniques.
Automatically generated - may contain errors
Lab products found in correlation
Eclipse ni u microscope, by Nikon (2 mentions)
Rabbit anti laminin, by Merck Group (1 mentions)
Rabbit anti pcna, by Cell Signaling Technology (1 mentions)
Nis elements analysis system, by Nikon (1 mentions)
Triton x 100, by Merck Group (1 mentions)
Lsm 780, by Nikon (1 mentions)
Propidium iodide, by Merck Group (1 mentions)
N acetyl d galactosamine, by Merck Group (1 mentions)
Bovine serum albumin (bsa), by Zhongshan Golden Bridge Biotechnology (1 mentions)
Nis software, by Nikon (1 mentions)
Schiff reagent, by Fujifilm (1 mentions)
5 protocols using biotinylated dba lectin
1
Immunohistochemical Analysis of Placental Tissue
2
Immunofluorescent Localization of CYP26A1 in Breast Cancer Cells
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Human breast cancer cell line (MCF‐7) cells, which express CYP26A1 protein, were cytospun onto slides. Then, the slides and sections (10 μm) of pregnant uteri were fixed in 4% PFA for 15 min. The slides were permeabilized in 0.3% Triton X‐100 (Sigma‐Aldrich) for 15 min. After being washed in PBS, the sections and the slides were blocked with 10% normal horse serum at 37°C for 1 hr. Then, the sections were incubated with biotinylated‐DBA lectin (Sigma‐Aldrich), and the slides were incubated with immunized mouse serum at 4°C overnight. Then, the sections were incubated with streptavidin‐FITC, and the slides were incubated with antimouse secondary antibody conjugated to FITC at 37°C for 1 hr. After staining, the sections and slides were washed in PBS and then mounted with antifade mounting media containing propidium iodide (Sigma‐Aldrich). Images were acquired using a confocal microscope (Zeiss LSM 780, Oberkhorn, Germany) or a Nikon ECLIPSE Ni‐U microscope (Nikon).
3
DBA Lectin Staining Protocol
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DBA lectin staining was performed as previously described (24 (link)). Endogenous peroxidase activity was blocked by incubating the sections in 3% H2O2 for 10 min at room temperature. Non-specific antibody binding was blocked in 5% bovine serum albumin (Zhongshan Golden Bridge Biotechnology Co., Ltd.) for 60 min at room temperature. The sections were then incubated in biotinylated-DBA lectin (dilution, 1:1,500; Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) overnight at 4°C. Following washing in PBS, the sections were incubated with streptavidin-peroxidase for another 60 min at 37°C. Positive signals were detected with 0.05% 3,3′-diaminobenzidine solution and counter-stained with 5% Harris' hematoxylin solution (all Zhongshan Golden Bridge Biotechnology Co., Ltd.) 2 min at room temperature. The addition of 0.1 M N-acetyl-D-galactosamine (Sigma-Aldrich; Merck KGaA) to the DBA lectin incubation provided a negative control.
4
Uterine Immunohistochemistry Protocol
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Anti-CX3CL1 primary Ab and biotinylated-DBA lectin (Sigma-Aldrich, St. Louis, MO, USA) were used. Cryosections (8 μm) of uteri were fixed in 4% PFA for 15 min. After being washed in PBS, the sections were blocked with 3% hydrogen peroxide for 5 min and sequential 10% horse normal serum (ZSGB-BIO, Beijing, China) at 37 °C for 1 h. Then, the cryosections were incubated with anti-CX3CL1 primary Ab or biotinylated-DBA lectin at 4 °C overnight, followed by incubation with secondary Ab conjugated to HRP or streptavidin-HRP (ZSGB-BIO) at 37 °C for 1 h.
The isolated uterine stromal cells were cultured for 12 h and fixed in 4% PFA for 15 min. After being washed in PBS, the cells were blocked with PBS containing 1% BSA at 37 °C for 1 h and then incubated with anti-CX3CL1 primary Ab at 4 °C overnight, followed by incubation with a secondary Ab conjugated to HRP at 37 °C for 1 h.
Cell and tissue slides were stained with diaminobenzidine (ZSGB-BIO) and counterstained with haematoxylin. Images were taken using a Nikon ECLIPSE Ni-U microscope and the NIS software (Nikon, Tokyo, Japan).
The isolated uterine stromal cells were cultured for 12 h and fixed in 4% PFA for 15 min. After being washed in PBS, the cells were blocked with PBS containing 1% BSA at 37 °C for 1 h and then incubated with anti-CX3CL1 primary Ab at 4 °C overnight, followed by incubation with a secondary Ab conjugated to HRP at 37 °C for 1 h.
Cell and tissue slides were stained with diaminobenzidine (ZSGB-BIO) and counterstained with haematoxylin. Images were taken using a Nikon ECLIPSE Ni-U microscope and the NIS software (Nikon, Tokyo, Japan).
5
Dual Histochemical Staining Protocol
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Formalin-fixed paraffin-embedded tissue sections (5 µm) were first stained with DBA lectin and then subjected to PAS staining. In brief, after antigen retrieval, sections were incubated with biotinylated DBA lectin (1:5000; Sigma) and visualized positive cells with DAB. Next, sections were incubated with 1% periodic acid (Fujifilm Wako Pure Chemical Corporation) for 20 min, with Schiff reagent (Fujifilm Wako Pure Chemical Corporation) for 30 min, and 0.5% sodium bisulfite solution (Fujifilm Wako Pure Chemical Corporation, Osaka, Japan) for 5 min. Hematoxylin was used for counter-staining.
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