Dako antigen retrieval solution

Manufactured by Agilent Technologies

Sourced in United States, Denmark

Dako antigen retrieval solution is a laboratory reagent designed to facilitate the process of antigen retrieval in immunohistochemistry and immunocytochemistry techniques. It is used to unmask or expose antigenic sites within fixed tissue or cell samples, which is a necessary step for effective antibody binding and subsequent detection.

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Lab products found in correlation

Lsm 780 confocal microscope, by Zeiss (3 mentions) Dako wash buffer, by Agilent Technologies (2 mentions) Permount, by Thermo Fisher Scientific (2 mentions) Rabbit anti iba1, by Proteintech (2 mentions) Mouse anti gfap, by Merck Group (2 mentions) Alexa fluor 555, by Thermo Fisher Scientific (2 mentions) Prolong gold antifade mountant with dapi, by Thermo Fisher Scientific (2 mentions) Zen blue software, by Zeiss (2 mentions) Lsm 880, by Zeiss (2 mentions) Mab377, by Merck Group (2 mentions) Anti rabbit antibody, by Thermo Fisher Scientific (2 mentions) Alexa fluor 568, by Thermo Fisher Scientific (2 mentions) Phosphohistone h3 ph3 ser10 antibody, by Cell Signaling Technology (1 mentions) Cleaved caspase 3 asp175 antibody, by Cell Signaling Technology (1 mentions) Leica autostainer xl, by Leica Biosystems (1 mentions) Axiocam, by Zeiss (1 mentions) Normal donkey serum (nds), by Jackson ImmunoResearch (1 mentions) Axioplan 2 microscope, by Zeiss (1 mentions) Dab substrate, by Vector Laboratories (1 mentions) Goat anti rabbit horseradish peroxidase secondary antibody, by Jackson ImmunoResearch (1 mentions)

25 protocols using dako antigen retrieval solution

Immunohistochemical Analysis of KS Lesions

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Immunohistochemical staining was performed on formalin-fixed paraffin-embedded biopsy specimens of human KS lesions (1 skin, 3 lymph nodes, and 1 lung) from the tissue repository of the Department of Pathology and Laboratory Medicine at the New York Presbiterian Hospital-Weill Cornell Medical Center. Immunohistochemical staining was performed on a TechMate 500 automated immunostainer (Ventana Medical Systems, Tucson AZ). For the COX-2 staining, sections were treated in a DAKO Antigen Retrieval Solution (DakoCytomation, Carpinteria, CA) and COX-2 was detected using an anti-human COX-2 antibody (Zymed Laboratories, San Francisco, CA) followed by an HRP-labelled secondary antibody detection system and DAB chromogen (DakoCytomation). For the second round of analysis by IHC, KS tissue sections were again retrieved with a DAKO Antigen Retrieval Solution (DakoCytomation) and KSHV-LANA was detected using a rat monoclonal to LANA-1 ORF 73 (Advanced Biotechnologies, MD), a secondary anti-rat biotinylated antibody (BD Pharmingen) and developed using an ABC Alkaline-Phosphatase complex (Ventana) and BT Red Reagent substrate (Ventana). Slides were counterstained with hematoxylin and mounted. Pictures were taken using an Olympus microscope equipped with a camera.

Medina M.V., D´Agostino A., Ma Q., Eroles P., Cavallin L., Chiozzini C., Sapochnik D., Cymeryng C., Hyjek E., Cesarman E., Naipauer J., Mesri E.A, & Coso O.A. (2020). KSHV G-protein coupled receptor vGPCR oncogenic signaling upregulation of Cyclooxygenase-2 expression mediates angiogenesis and tumorigenesis in Kaposi’s sarcoma. PLoS Pathogens, 16(10), e1009006.

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Quantifying Neuroepithelial Proliferation and Apoptosis

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Three control and three NTD embryos on gestation day 11.5 formalin-fixed embryos were paraffin-embedded and cut into 5 μm sections. The sections were dried on a slide dryer at 58 °C for 1 h, then dewaxed and rehydraed by a series of xylol and ethanol rinses using the Leica Autostainer XL (Leica Biosystems Nussloch GmbH, Germany). The Dako antigen retrieval solution was used for heat-activated antigen retrieval (pH 6.0) (Dako, Glostrup, Denmark). The phosphohistone H3 (PH3) (Ser10) antibody (Cell Signaling, 1:400) and the cleaved caspase-3 (Asp175) antibody (Cell Signaling, Boston, MA, USA; 1: 250) were uesd to detect the proliferation and apoptosis of neuroepithelial cells in the sections. Six equal-sized fields were randomly selected and the mean number of positive cells was counted under light microscope.

Xu L., Wang L., Wang J., Zhu Z., Chang G., Guo Y., Tian X, & Niu B. (2016). The effect of inhibiting glycinamide ribonucleotide formyl transferase on the development of neural tube in mice. Nutrition & Metabolism, 13(1), 56.

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RA Synovial Tissue RANKL Expression

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RA synovial tissue biopsy samples (26 (link)) were fixed in neutral buffered formalin and embedded in paraffin. Slides were incubated at 60°C for 1 hour, transferred to xylene, and hydrated by sequential immersion in ethanol, 95% ethanol, 75% ethanol, and, finally, water. Antigens were unmasked in Dako antigen retrieval solution (S1699) for 30 minutes. Nonspecific binding was blocked with 5% normal donkey serum (017-000-121; Jackson ImmunoResearch) for 30 minutes at room temperature. Primary antibodies were added to the slides and incubated at room temperature in a humid chamber overnight, followed by secondary antibodies for 2 hours at room temperature. Tissues were washed in PBS and mounted with ProLong Gold Antifade Mountant with DAPI (P-36931; Life Technologies). Photomicrographs were obtained with a Zeiss Axioplan 2 microscope and recorded with a Zeiss AxioCam digital camera.
Positivity for RANKL within the CD20+ B cell and CD3+ T cell populations was determined, and CD20+ B cells expressing RANKL were quantitated. T and B cells were enumerated in 8 random 200 × fields using an automated tool in Zeiss Axioplan software, and the percentages of RANKL-expressing cells were calculated. B cells positive for RANKL were enumerated in 7 random 200 × fields, and the percentages of neighboring cells were calculated.

Meednu N., Zhang H., Owen T., Sun W., Wang V., Cistrone C., Rangel-Moreno J., Xing L, & Anolik J.H. (2016). Production of RANKL by Memory B Cells: A Link Between B Cells and Bone Erosion in Rheumatoid Arthritis. Arthritis & rheumatology (Hoboken, N.J.), 68(4), 805-816.

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Immunohistochemical Analysis of Mouse Tissue

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Unstained sections of mouse tissue were deparaffinized and rehydrated. Antigen retrieval was performed using Dako antigen retrieval solution (Dako North America, Carpinteria, CA). Endogenous peroxidase was blocked using 3% hydrogen peroxide. Tissues were incubated with primary antibodies against Ki67 (Thermo/Lab Vision Corp., Fremont, CA) and CD31 (Abcam, Cambridge, MA) overnight at 4°C. Next day, a goat anti-rabbit horseradish peroxidase secondary antibody (Jackson ImmunoResearch Laboratories, West Grove, PA) diluted in blocking solution was added and the samples were incubated for 1 h at room temperature. Slides were developed with DAB substrate (Vector Laboratories, Inc., Burlingame, CA) and counterstained with Gill no. 3 hematoxylin solution. Positive (DAB-stained) cells were counted in five random fields per slide.

Kanlikilicer P., Rashed M.H., Bayraktar R., Mitra R., Ivan C., Aslan B., Zhang X., Filant J., Silva A.M., Rodriguez-Aguayo C., Bayraktar E., Pichler M., Ozpolat B., Calin G.A., Sood A.K, & Lopez-Berestein G. (2016). Ubiquitous Release Of Exosomal Tumor Suppressor miR-6126 from Ovarian Cancer Cells. Cancer research, 76(24), 7194-7207.

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Immunohistochemical Evaluation of CMPK1 in Breast Cancer

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Sections of 4 μm of the three above mentioned TMAs were incubated for 1 hour at room temperature with CMPK1 antibody at a dilution of 1:300 against the protein (mouse monoclonal, clone 1D7; Lifespan Bioscience, Seattle, WA, USA). Antigen retrieval was performed prior to antibody incubation by heating the slides for 40 min at 95 °C and washing with Dako antigen retrieval solution (pH = 6) (DakoCytomation, Carpinteria, CA, USA) when the slides were cooled down to room temperature. Staining was visualized by anti-mouse EnVision+^® System-HRP (DAB) (DakoCytomation, Carpinteria, CA, USA). CMPK1 staining was separately scored in both percentage of positive invasive breast carcinoma cells and staining intensity by two independent observers. Six categories were scored for the percentage of positive invasive tumor cells: 0%, 1–10%, 11–25%, 26–50%, 51–70%, and >70%. All cores present on the three TMAs were scored by an experienced researcher in a blind manner. Staining score of triplicate cores were validated by a second experienced researcher, who was extensively trained by a specialized breast pathologist.

Liu N.Q., De Marchi T., Timmermans A., Trapman-Jansen A.M., Foekens R., Look M.P., Smid M., van Deurzen C.H., Span P.N., Sweep F.C., Brask J.B., Timmermans-Wielenga V., Foekens J.A., Martens J.W, & Umar A. (2016). Prognostic significance of nuclear expression of UMP-CMP kinase in triple negative breast cancer patients. Scientific Reports, 6, 32027.

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Apoptosis Detection in Smooth Muscle Cells

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SMCs were treated with staurosporine (50 nM; Sigma) for 18 h to induce apoptosis. ApopTag apoptosis detection kit (TUNEL; Merck Millipore, Darmstadt, Germany) was then used according to manufacturer’s instructions to label apoptotic cells. Similarly, tissue sections, processed as described in “Immunofluorescence staining”, underwent heat-induced antigen retrieval with Dako antigen retrieval solution (Dako) in a streamer for 20 min, followed by blocking with Image-iT FX signal enhancer (Invitrogen) for 30 min. Staining was performed according to manufacturer’s instructions using the ApopTag apoptosis detection kit. Nuclei were counterstained with DAPI. The percentage of TUNEL-positive cells was calculated.

Deuse T., Hua X., Wang D., Maegdefessel L., Heeren J., Scheja L., Bolaños J.P., Rakovic A., Spin J.M., Stubbendorff M., Ikeno F., Länger F., Zeller T., Schulte-Uentrop L., Stoehr A., Itagaki R., Haddad F., Eschenhagen T., Blankenberg S., Kiefmann R., Reichenspurner H., Velden J., Klein C., Yeung A., Robbins R.C., Tsao P.S, & Schrepfer S. (2014). Dichloroacetate prevents restenosis in preclinical animal models of vessel injury. Nature, 509(7502), 641-644.

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Immunohistochemistry for CD34 and Collagen 1

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The tissue biopsies were embedded in paraffin wax and sectioned into 5 µm sections. We followed the usual immunohistochemistry procedure with antigen retrieval for 30 min in Dako antigen retrieval solution (Dako, UK) at 95°C. After washing with PBS tissue sections were incubated in blocking buffer (PBS+2% FBS) for half an hour and then incubated in primary mouse anti human CD34 and rabbit anti-human Collagen 1 (1∶100 dilution) antibodies (Abcam, UK) overnight at 4°C. Secondary anti-mouse FITC conjugated (1∶250 dilution) and anti-rabbit AF-546 conjugated (1∶250 dilution) antibodies (Jackson laboratories, US) were added to the sections after thorough washing with PBS and incubated for an hour at room temperature. DAPI was added to the tissue section for nuclei staining. Isotype matched antibodies were used as a negative control.

Iqbal S.A., Hayton M.J., Watson J.S., Szczypa P, & Bayat A. (2014). First Identification of Resident and Circulating Fibrocytes in Dupuytren’s Disease Shown to Be Inhibited by Serum Amyloid P and Xiapex. PLoS ONE, 9(6), e99967.

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Immunofluorescence Staining Protocol

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Following deparaffinization in xylene and rehydration in descending alcohol series, heat-mediated antigen retrieval was performed by incubating hydrogel sections in Dako antigen retrieval solution (Dako) for 20 min at 98 °C. This step was followed by cell permeabilization with 0.01% (v/v) Triton-X (Sigma-Aldrich) solution in PBS, for 20 min. Samples were then incubated with 5% (v/v) Goat or Donkey Serum (Dako) for 30 min. Primary antibodies diluted in Dako antibody diluent solution, were incubated overnight at 4 °C, followed by washing steps and incubation with Alexa-coupled secondary antibody (1:500) and Hoechst 33412 (1:500) solution for 1 h at room temperature. Finally, samples were washed in PBS, and mounted with Vectashield antifade mounting medium (Vector Laboratories). Stained sections were imaged using laser scanning confocal microscope (LSM 880, Zeiss, Germany), and image processing were performed on ZEN Blue software (Zeiss, Germany).

Grebenyuk S., Abdel Fattah A.R., Kumar M., Toprakhisar B., Rustandi G., Vananroye A., Salmon I., Verfaillie C., Grillo M, & Ranga A. (2023). Large-scale perfused tissues via synthetic 3D soft microfluidics. Nature Communications, 14, 193.

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Immunohistochemistry for BrdU and Caspase-3

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After deparaffinization, the slides were heated in the microwave in Dako antigen retrieval solution ((S1699), Dako, Carpinteria, CA, USA) and then washed in Dako wash buffer (S3006). Sections were blocked with 0.5% BSA in PBS for 30 min at room temperature followed by Dako peroxidase block for 30 min. Primary anti-BrdU (Bromo-deoxyuridine) (Millipore, Burlington MA, USA) or anti-Cleaved Caspase 3 antibody (Cell Signaling, Danvers, MA, USA) was placed on sections at a concentration of 1:200 at room temperature for 1 h or overnight at 4 °C. Sections were then treated with Dako horseradish peroxidase (HRP)-linked secondary antibody (anti-rabbit or mouse IgG from Dako, Carpinteria, CA, USA) for 30 min at room temperature and developed with 3,3′-Diaminobenzidine (DAB) reagent. Immunohistochemistry for BrdU and caspase 3 were independently evaluated manually by two investigators (CT and GM), verified by ImageJ software (National Institute of Health, Bethesda, Rockville, MD, USA), and calculated as an average of the percentages of positive nuclei per total number of nuclei per field of view for each lesion evaluated.

Ding Y., Mullapudi B., Torres C., Mascariñas E., Mancinelli G., Diaz A.M., McKinney R., Barron M., Schultz M., Heiferman M., Wojtanek M., Adrian K., DeCant B., Rao S., Ouellette M., Tsao M.S., Bentrem D.J, & Grippo P.J. (2018). Omega-3 Fatty Acids Prevent Early Pancreatic Carcinogenesis via Repression of the AKT Pathway. Nutrients, 10(9), 1289.

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Immunofluorescence Staining of Brain Tissue

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To retrieve antigens with a DAKO antigen retrieval solution (DAKO, Carpenteria, CA, USA), brain samples were heated by steam for 30 min. To inhibit endogenous peroxidase, a series of slices were washed with Tris-buffered saline (TBS) and immersed in a 3% hydrogen peroxide solution for 10 min. Slices were then incubated with a TUNEL kit and primary antibodies (mouse anti-NeuN antibodies (Merck; MAB377; Darmstadt, Germany); rabbit anti-Iba1 (proteintech; 10904-1-AP; Rosemont, IL, USA); and mouse anti-GFAP antibodies (Sigma; G3893; USA)) at room temperature. After being washed twice with TBS, the slices were incubated with anti-mouse antibodies (Thermo; A10524; Waltham, MA, USA) and anti-rabbit antibodies (Thermo; A11008; Waltham, MA, USA) at room temperature for 3 h. Next, the slices were stained and mounted within Fluroshield TM with DAPI (Sigma; F6057; USA) after being washed twice with TBS. Images were captured with a fluorescence microscope (Olympus, U-RFL-T) and the fluorescence intensity was measured by Image J vers. 1.44d software (NIH).

Wu C.H., Tsai H.P., Su Y.F., Tsai C.Y., Lu Y.Y, & Lin C.L. (2022). 2-PMAP Ameliorates Cerebral Vasospasm and Brain Injury after Subarachnoid Hemorrhage by Regulating Neuro-Inflammation in Rats. Cells, 11(2), 242.

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