Dako serum free blocking solution

Manufactured by Agilent Technologies

Sourced in United States

Dako serum-free blocking solution is a laboratory reagent designed to block non-specific binding in immunohistochemical and in situ hybridization procedures. It is a ready-to-use solution that can be applied directly to tissue sections or cells to reduce background staining and improve signal-to-noise ratio.

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Lab products found in correlation

Axio imager m1 microscope, by Zeiss (3 mentions) Ab14106, by Abcam (2 mentions) Ab94744, by Abcam (1 mentions) Diaminobenzidine substrate chromogen system, by Agilent Technologies (1 mentions) Avidin biotinylated enzyme complex, by Vector Laboratories (1 mentions) Dp260, by Olympus (1 mentions) Ab14933, by Abcam (1 mentions) Alexa fluor 488 or 546, by Thermo Fisher Scientific (1 mentions) Ab91654, by Abcam (1 mentions) 4 well cell culture slides, by MatTek (1 mentions) Anti pcna, by Santa Cruz Biotechnology (1 mentions) Anti sm α actin, by Agilent Technologies (1 mentions) Anti mac2, by Cedarlane (1 mentions) Vectastain abc hrp kit, by Vector Laboratories (1 mentions) Dab peroxidase substrate kit, by Vector Laboratories (1 mentions) Avidin biotinylated enzyme complex abc, by Vector Laboratories (1 mentions) Anti cd31 antibody, by Santa Cruz Biotechnology (1 mentions)

Close spelling variants of this product

Serum-free blocking solution DAKO blocking solution Blocking solution

5 protocols using dako serum free blocking solution

Histological Analysis of Aortic Calcification

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After organ culture for 7 days, thoracic aorta segments were washed with PBS and fixed with 10% neutral buffer formalin. The specimens were then embedded in paraffin, and 5μm cross-sections were cut. The sections were deparaffinized and H&E staining was performed. Arterial medial calcification was visualized using Alizarin Red S staining as previously described ^{23 (link)}. For immunohistochemistry, the sections were deparaffinized, followed by treatment with citrate buffer for antigen retrieval and 3% H₂O₂. The sections were blocked with Dako serum-free blocking solution (Dako North America, USA) and incubated with primary antibody for overnight at 4°C. The primary antibodies included SM22α (Abcam, ab14106), Osterix (Abcam, ab94744). Subsequently, the sections were incubated with biotinylated secondary antibodies for 30 min at room temperature. Avidin-biotinylated enzyme complex (Vector Laboratories, USA) and a diaminobenzidine substrate chromogen system (Dako North America, USA) were used for detection. Sections were counterstained with hematoxylin. The images were captured by Olympus DP260.

Lin T., Wang X.L., Zettervall S.L., Cai Y, & Guzman R.J. (2016). DMH1, a Highly Selective Small Molecule BMP Inhibitor, Suppresses Arterial Medial Calcification. Journal of vascular surgery, 66(2), 586-593.

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Immunofluorescent Analysis of Aortic Tissue

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Aortas isolated from vehicle or vitamin D₃-treated rats were fixed with 10% NBF and embedded in paraffin. The sections were deparaffinized and treated with 10 mM HIER citrate buffer (pH 6.0) for antigen retrieval, and then blocked with Dako serum-free blocking solution (Dako), followed by incubation of secondary antibody Alexa Fluor 488 or 546 (Thermo Fisher Scientific). The antibodies including anti-LAMP2 (Santa Cruz Biotechnology, sc-20004), anti-RUNX2 (BML Life science, D130-3), anti-BMP2 (Abcam, ab14933), and anti-SM22α (Abcam, ab14106) were used. Nuclei were stained with DAPI. Cells were visualized with Zeiss AxioImager M1 microscope.

Cai Y., Wang X.L., Flores A.M., Lin T, & Guzman R.J. (2018). Inhibition of endo-lysosomal function exacerbates vascular calcification. Scientific Reports, 8, 3377.

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Immunofluorescent Staining of SMCs

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SMCs were seeded on 4-well cell culture slides (MatTek), fixed in 4% PFA, permeabilized in 0.1% Triton x-100/PBS, and blocked with Dako serum-free blocking solution (Dako, X090930). The immunofluorescent staining was conducted using the primary antibody, followed by incubation of secondary antibody Alexa Fluor 546 or 488 (ThermoFisher Scientific). The primary antibodies against BrdU (Bio-Rad, MCA2483T) and CRTC3 (Abcam, ab91654) were used. Nuclei were stained with DAPI. The staining was visualized with Zeiss AxioImager M1 microscope.

Cai Y., Wang X.L., Lu J., Lin X., Dong J, & Guzman R.J. (2021). Salt-inducible kinase 3 promotes vascular smooth muscle cell proliferation and arterial restenosis by regulating AKT and PKA-CREB signaling. Arteriosclerosis, thrombosis, and vascular biology, 41(9), 2431-2451.

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Immunohistochemical Analysis of Femoral Arteries

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Femoral arteries were dissected and embedded in paraffin, and cross-sections were cut at 200 μm intervals. The sections were deparaffinized and treated with citrate buffer for antigen retrieval, followed by incubation in 3% H₂O₂. The sections were then blocked with Dako serum-free blocking solution (Dako, X090930) and incubated with primary antibody at 4°C overnight. Next, the sections were incubated with biotinylated secondary antibodies for 30 min at room temperature. VECTASTAIN ABC HRP kit (Vector Laboratories, PK-4000) and DAB peroxidase substrate kit (Vector Laboratories, SK-4100) were used for detection. Hematoxylin was used for nuclear counterstaining. The primary antibodies used in this study are anti-SIK3 (LSBio, LS-B9603), anti-SM-α-actin (Dako, M0851), anti-PCNA (Santa Cruz, sc-56), and anti-Mac2 (Cedarlane, CL8942AP). The matched IgG was used for negative control. Slides were visualized, and images were captured by Zeiss AxioImager M1 microscope.

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Histological Evaluation of Muscle and Arterial Changes

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Gastrocnemius muscle and femoral arteries from VitD₃ or vehicle‐treated rats were fixed with 10% neutral buffered formalin (10% NBF). The tissues were embedded in paraffin, sectioned, and deparaffinized. Von kossa staining was used to assess calcification. H&E staining was used to evaluate the muscle fiber morphology. Verhoeff–van Gieson (VVG) staining was used to examine the integrity of elastic fibers in arteries.
To determine capillary density, CD31 immunohistochemistry staining was performed. After deparaffinized, gastrocnemius muscle sections were treated with 10 mM HIER citrate buffer (pH 6.0) for antigen retrieval and then blocked with Dako serum‐free blocking solution (Dako) followed by incubation of anti‐CD31 antibody (Santa Cruz, sc‐1506) and biotinylated anti‐goat secondary antibody. Avidin‐biotinylated enzyme complex (ABC) (Vector Laboratories) and a diaminobenzidine (DAB) substrate chromogen system (Dako) were used to detect CD31. The sections were counterstained with hematoxylin. Slides were viewed with an Olympus microscope with a digital camera. Capillaries and muscle fibers were counted manually by a blinded observer using ImageJ software in 10 different randomized fields per slide (40×) and shown as densities per high power field.

Zettervall S.L., Wang X., Monk S., Lin T., Cai Y, & Guzman R.J. (2021). Recovery of limb perfusion and function after hindlimb ischemia is impaired by arterial calcification. Physiological Reports, 9(16), e15008.

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