Dako liquid dab

Manufactured by Agilent Technologies

Sourced in Australia, United States

Dako Liquid DAB is a chromogen solution used in immunohistochemistry (IHC) and in situ hybridization (ISH) procedures. It provides a brown, visible reaction product at the site of the target antigen or nucleic acid sequence.

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4 protocols using dako liquid dab

Quantifying DNA Damage by N7-Methylguanine Immunohistochemistry

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DNA damage was measured by quantitative immunohistochemistry using a rabbit polyclonal antibody to N⁷-methylguanine (gift from Dr. Geoff Margison, Paterson Institute for Cancer Research, Manchester, UK). Tissue sections were placed in prewarmed 50 mM NaOH/40% ethanol at 55 °C to denature DNA and neutralized with 5 % acetic acid/40 % ethanol. Sections were incubated with the primary antibody followed by biotinylated goat anti-rabbit IgG. The antibody-antigen complex was visualized using the DAKO Liquid DAB (diamino-benzidine tetrahydrochloride) Substrate-Chromagen System (DAKO, Carpinteria, CA). Liver N⁷-methylguanine DNA adducts in AOM-injected animals were used as a positive control. Omission of primary antibody was used as a negative control. Images of colonic crypts were visualized on a MICROSTAR IV, Reichert light microscope, captured by a digital camera (Sony DXC-970 MD, color 3CCD) and staining intensity (assessed by cell position) was analyzed using NIH Image software (NIH Image, version 1.61). Each nucleus on one side of a crypt column was outlined and the staining intensity was measured. Background staining intensity was subtracted from the staining intensity of the nuclei.

Hong M.Y., Turner N.D., Murphy M.E., Carroll R.J., Chapkin R.S, & Lupton J.R. (2015). In Vivo Regulation of Colonic Cell Proliferation, Differentiation, Apoptosis and P27Kip1 by Dietary Fish Oil and Butyrate in Rats. Cancer prevention research (Philadelphia, Pa.), 8(11), 1076-1083.

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Immunohistochemical Staining of CD3+ T Cells

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Ear sections were deparaffinized and rehydrated using the Leica ST5020. Samples underwent heat-induced epitope retrieval, in sodium citrate, pH 6.0 at 95ºC for 20 minutes. The sections were then blocked for non-specific enzymatic reactions with Dual Endogenous Enzyme Block (cat. # S2003, Dako) for 10 minutes at room temperature. This was followed by blocking for non-specific protein interactions with Protein Block Serum-free (cat. # X0909, Dako) for 20 minutes at room temperature. Subsequent staining and detection with polyclonal rabbit anti-human CD3 (cat. # A0452, Dako) for 1 hour, followed by Dako EnVision+ system-HRP anti-rabbit polymer (cat. # K4002, Dako) for 30 minutes was performed. DAKO® Liquid DAB (3,3′diaminobenzidine tetrahydrochloride) + Substrate-Chromogen System (cat. # K3467, Dako) was used for 3 minutes, then counterstained for 1 minute with Gills hematoxylin for visualization of antigen-antibody reactions. Sections were then dehydrated on the Leica ST5020 Multistainer and coverslipped using a Leica CV5030. A USDA pathologist, who was blinded to the treatment groups, evaluated and scored the slides.

Putz E.J., Putz A.M., Boettcher A., Charley S., Sauer M., Palmer M., Phillips R., Hostetter J., Loving C.L., Cunnick J.E, & Tuggle C.K. (2019). Successful development of methodology for detection of hapten-specific contact hypersensitivity (CHS) memory in swine. PLoS ONE, 14(10), e0223483.

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Nuclear Factor-kappa B Activation Assay

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BMMs were seeded into 96-well plates at the density of 2 × 10⁴/well and then incubated at 37 °C overnight. The next day, cells were first pre-incubated with NC (1 μM) for 1 h, then stimulated with RANKL (100 ng/mL) for 30 min. The cells were then washed once with 1 × PBS, then fixed with 4% PFA for 10 min. After washed three more times with 1 × PBS, cells were permeablized for 5 min with 0.1% Triton X-100 in PBS. After that, cells were washed twice with 0.1% BSA-PBS, cells were incubated at 37 °C for 45 min with anti-p65 antibody (50 μl/well; Santa Cruz Biotechnology, Inc., CA, USA) diluted 1:200 in 0.1% BSA-PBS. The cells were then washed four times with 0.1% BSA-PBS, four times with 1 × PBS, once with 0.1% BSA-PBS before the addition of streptavidin-horseradish peroxidase (Dako, Victoria, Australia). After incubation for 20 min at room temperature and washing for four times with 0.1% BSA-PBS, four times with PBS, once with 0.1% BSA-PBS Dako Liquid DAB (20 μL/well; Dako, Victoria, Australia) was added to the plates for up to 30 min or until brown color presented.

Liu Q., Wang T., Zhou L., Song F., Qin A., Feng H.T., Lin X.X., Lin Z., Yuan J.B., Tickner J., Liu H.G., Zheng M.H., Xu J, & Zhao J.M. (2016). Nitidine chloride prevents OVX-induced bone loss via suppressing NFATc1-mediated osteoclast differentiation. Scientific Reports, 6, 36662.

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Luteoloside Modulates Osteoclastogenesis

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Alpha modified Minimal Essential Medium (α-MEM) and fetal bovine serum (FBS) were obtained from Thermo Fisher Scientific (Scoresby, Vic, Australia). Luteoloside of purity >95% was purchased from Chengdu Must Bio-Technology Co., Ltd (Chengdu, Sichuan Province, China) and dissolved in Dimethyl sulfoxide (DMSO) at a concentration of 100 μM. Anti-ERK, anti-phospho-ERK, anti-JNK, anti-phospho-JNK, anti-p38, anti-phospho-p38, anti-IκBα, anti-p65, anti-NFATc1, anti-c-Fos and anti-ß-Actin antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Vacuolar-type H+-ATPase V0 subunit d2 (V-ATPase d2) was generated as previously described [15] . The MTS assay kit and luciferase assay system were obtained from Promega (Madison, WI, USA). Leucocyte acid phosphatase staining kits were obtained from Sigma-Aldrich (St Louis, MO, USA).
Recombinant macrophage colony stimulating factor (M-CSF) was obtained from R&D Systems (Minneapolis, MN, USA). Recombinant GST-rRANKL protein was expressed and purified as previously described [16] . Streptavidin-horseradish peroxidase and Dako Liquid DAB were purchased from DAKO (Carpinteria, CA, USA,). ProLong Diamond anti-fade mounting medium were obtained from Invitrogen

Song F., Wei C., Zhou L., Qin A., Yang M., Tickner J., Huang Y., Zhao J, & Xu J. (2018). Luteoloside prevents lipopolysaccharide-induced osteolysis and suppresses RANKL-induced osteoclastogenesis through attenuating RANKL signaling cascades. Journal of cellular physiology, 233(2).

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