Diva decloaker solution

Manufactured by Biocare Medical

Sourced in United States

Diva Decloaker solution is a laboratory reagent used for heat-induced epitope retrieval (HIER) during immunohistochemical (IHC) staining procedures. It is designed to aid in the unmasking of antigenic sites within formalin-fixed, paraffin-embedded tissue sections prior to the application of primary antibodies.

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9 protocols using diva decloaker solution

Immunofluorescence Analysis of Renal Tissue

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FFPE mouse renal (4 μm) sections were deparaffinized using xylene followed by graded ethanols for rehydration according to the standard protocol. Antigen retrieval was performed using Diva Decloaker solution (Biocare Medical, Concord, CA). After being blocked with blocking buffer containing 5% BSA in PBS-T, the sections were incubated over night, 4 °C with Lotus tetragonolobus agglutinin (LTA) (FL-1321, Vector laboratories, Burlingame, CA) and CaIX (AF2344, R&D Systems, Minneapolis, MN). After washing, secondary antibody donkey -anti rabbit AlexaFluor-594 (A-11056, Life technologies, Carlsbad, CA) and nuclear stain To-pro-3 iodide (Life technologies, Carlsbad, CA) were applied according to the manufacturer’s instructions followed by rinsing and mounting. Sections were subsequently analyzed by confocal scanning using the Zeiss LSM 710 system (Carl Zeiss AG, Oberkochen, Germany).

Johansson E., Rönö B., Johansson M., Lindgren D., Möller C., Axelson H, & Smith E.M. (2016). Simultaneous targeted activation of Notch1 and Vhl-disruption in the kidney proximal epithelial tubular cells in mice. Scientific Reports, 6, 30739.

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Immunofluorescence Staining for Neural Markers

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All immunofluorescence was carried out on slide-mounted sections. In brief, sections were lightly fixed for 10 min at RT in 4% PFA/PBS, then washed in PBS for three times for 5 min each. Heat-induced epitope retrieval was performed using a Biocare Medical Decloaking Chamber NxGen using the DIVA Decloaker solution (Biocare Medical) diluted at 1× working concentration in deionized water. Samples were washed in PBS and incubated with 10% donkey serum in PBS + 0.1% Triton X-100 to permeabilize the tissue and prevent nonspecific antibody binding. Primary antibody was applied and incubated overnight at 4°C. The following primary antibodies and respective dilutions were used: Tbr1 (Abcam, ab31940), 1:200; Ctip2 (Abcam, ab18465), 1:200; Brn2 1:200; Pax6 (Covance PRB-278P-100), 1:100; Tbr2 (Abcam, ab183991), 1:100; Ki67 (Abcam, ab15580), 1:200.

Harlan De Crescenzo A., Panoutsopoulos A.A., Tat L., Schaaf Z., Racherla S., Henderson L., Leung K.Y., Greene N.D., Green R, & Zarbalis K.S. (2020). Deficient or Excess Folic Acid Supply During Pregnancy Alter Cortical Neurodevelopment in Mouse Offspring. Cerebral Cortex (New York, NY), 31(1), 635-649.

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Immunohistochemical Identification of Immune Cell Subsets in Breast Cancer Sentinel Lymph Nodes

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Archived or fresh formalin-fixed paraffin-embedded biopsies of SLNs from breast cancer patients were sectioned and affixed to microscope slides. They were deparaffinized with xylene and rehydrated with decreasing concentrations of ethanol in water. Antigen retrieval was performed in a Digital Decloaking Chamber in DIVA Decloaker solution (Biocare Medical, Concord, California, USA). Tissue was stained with the following purified primary antibodies: mouse anti-human Cytokeratin (clone AE1/AE3; Biocare Medical); mouse anti-human CD20 (clone L26; Biocare Medical); rabbit anti-human CD3 (clone SP7; Biocare Medical), and mouse anti-human CD1a (clone CD1a007; Biocare Medical). Slides were subsequently stained with IgG secondary antibodies conjugated to alkaline phosphatase or horseradish peroxidase polymers. The antibody complexes were developed with diaminobenzidine (Biocare Medical), fast red (Biocare Medical), fast blue (Biocare Medical), NBT-BCIP (DAKO, Carpinteria, CA, USA), or VIP (Vector Laboratories, Burlingame, CA, USA). The cell nucleus was stained with hematoxylin (Biocare Medical).

Blenman K.R., He T.F., Frankel P.H., Ruel N.H., Schwartz E.J., Krag D.N., Tan L.K., Yim J.H., Mortimer J.E., Yuan Y, & Lee P.P. (2018). Sentinel lymph node B cells can predict disease-free survival in breast cancer patients. NPJ Breast Cancer, 4, 28.

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Immunohistochemical Tumor Characterization

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Histological sections of tumor tissues were analyzed following H&E staining and additional immunostaining on selected sections to determine cell origins. IHC was performed on 4 μm slides of formalin-fixed and paraffin-embedded tumor tissue specimens. The sections were deparaffinized with changes of xylenes and graded ethanols to water. Antigen retrieval was performed in a Decloaking Chamber (Biocare) with Diva DeCloaker solution (Biocare Medical, DV 2004). Staining procedures were performed on a Biocare IntelliPath autostainer. Blocking was performed using Peroxidazed 1 (PX968, Biocare Medical) for 5 min, and Background Punisher (BP974, Biocare Medical) for 10 min. Sections were then incubated for 30 min at room temperature with a prediluted monoclonal antibody (Vimentin, SM Actin, MS Actin or Cytokeratin; Biocare Medical). Secondary antibody, mouse on canine polymer (Biocare Medical, MC541) was applied for 30 min. Finally, the chromagen, diaminobenzidine (DAB) was incubated for 5 min (IP FLX DAB, IPK5010, Biocare Medical) and followed by hematoxylin counterstain (CATHE, Biocare Medical) for 2 min. Negative controls were obtained by replacing the primary antibody with Polymer negative control serum (Biocare Medical, NC499) for 30 min.

Schook L.B., Collares T.V., Hu W., Liang Y., Rodrigues F.M., Rund L.A., Schachtschneider K.M., Seixas F.K., Singh K., Wells K.D., Walters E.M., Prather R.S, & Counter C.M. (2015). A Genetic Porcine Model of Cancer. PLoS ONE, 10(7), e0128864.

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Correlative Light-Electron Microscopy of Lung Tissue

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Epitope retrieval from paraffin-embedded 5 µm sections of lung tissue was performed by incubating the slides in DIVA decloaker solution (Biocare Medical) for 40 min at 100°C, followed by Hot Rinse solution (Biocare Medical) for 5 min and phosphate-buffered saline (PBS) for 5 min. The sections were incubated with serum-free protein block (Dako) for 5 min and then with anti-collagen XIII antibody (Atlas Antibodies Cat# HPA050392) diluted 1:100 in PBS overnight. After three washes with PBS, the sections were incubated with Cy3 Goat Anti-Rabbit IgG (Jackson ImmunoResearch Labs Cat# 111-165-144) diluted 1:300 in PBS for 30 min and again washed three times with PBS. The coverslips were mounted with Immu-Mount (Thermo Scientific). The samples were imaged with a Leica SP8 Falcon confocal microscope with only individual optical sections used. After imaging, the coverslip was detached by incubating in PBS. The sections were postfixed with 1% osmium tetroxide, dehydrated in acetone and embedded in Epon LX112 using gelatin capsules. The region of interest was identified microscopically, trimmed and thin sections were prepared. The samples were imaged with a Tecnai G2 Spirit electron microscope. The light and electron microscopy images were correlated using nuclear staining with the eC-CLEM plugin33 (link) for Icy.34 (link)

Norman O., Koivunen J., Kaarteenaho R., Salo A.M., Mäki J.M., Myllyharju J., Pihlajaniemi T, & Heikkinen A. (2023). Contribution of collagen XIII to lung function and development of pulmonary fibrosis. BMJ Open Respiratory Research, 10(1), e001850.

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Vocal Fold Mucin Immunohistochemistry

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For all vocal fold mucin experiments vocal folds were tested within 1.5 hours of animal sacrifice. Primary and secondary antibodies for MUC1, MUC4, and MUC5AC are listed in Table 1. Positive controls included porcine lung and intestine (MUC1, MUC4) and rat intestine (MUC5AC). Vocal folds (N=3) were fixed in 10% neutral buffered formalin, embedded in paraffin, and sectioned (5μm). Sections were heat-retrieved using either Diva Decloaker solution (Biocare Medical, Concord, CA) or citrate buffer (pH6). Blocking was performed at room temperature by incubating sections with 3% H₂O₂ for 10 minutes and then either 2% horse serum for 20 minutes or 10% bovine serum albumin for 60 minutes. Primary antibodies were applied for 60 minutes at room temperature with biotinylated secondary antibodies added for 30 minutes. Visualization was achieved using a 3′Diaminobenzidine (DAB) horseradish peroxidase substrate kit for immunohistochemistry for 3–5 minutes (BD Biosciences, San Diego, CA). Sections were rated for positive and negative staining by a blinded observer familiar with vocal fold structure and immunohistochemical staining techniques. Sections were viewed and photographed using a Nikon Eclipse E600 microscope (Tokyo, Japan) with associated Olympus DP71 digital camera (Center Valley, PA).

Levendoski E.E, & Sivasankar M.P. (2014). Vocal fold ion transport and mucin expression following acrolein exposure. The Journal of membrane biology, 247(5), 441-450.

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Quantifying Immune and Vascular Markers in Liver and Intestine

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Left lobes of liver tissues and small intestines were excised and fixed in 4% paraformaldehyde (Santa Cruz Biotechnology) overnight at 4°C. Ten-micron paraffin-embedded sections were prepared and slides were boiled in Diva Decloaker solution (Biocare Medical; #DV2004) in a pressurized chamber for 15 min. Sections were blocked with in PBS containing 5% donkey serum, 1% bovine serum albumin (BSA) (Sigma-Aldrich) and 0.03% Triton-X (Plusone, #17-1315-01) for 1 hour, then incubated in with rat anti-F4/80 (Abcam, #ab6640), goat anti-S100A9 (R&D Systems, #AF2065), rabbit anti-von Willebrand Factor (DAKO, #a0082), rat anti-PV1 (BD pharmingen, #550563), or rabbit anti-ABCA1 (Novus Biologicals, NB400-105) at 4°C overnight. Primary antibodies were detected using Cy3- or Cy5-conjugated secondary antibodies (Jackson ImmunoResearch). The stained sections were imaged using an SP8 confocal microscope (Leica) equipped with nine lasers and four tunable detectors (two hybrid, two tunable) and a 20X HC PL Apo CS 2 multi-immersion objective, NA 0.75. Images were processed with Imaris software (Bitplane). Ten fields were quantified and averaged for each sample, with cell counts per image quantified using Image J software (NIH). All slides were analyzed by in blinded and randomized fashion.

Han Y.H., Onufer E.J., Huang L.H., Sprung R.W., Davidson W.S., Czepielewski R.S., Wohltmann M., Sorci-Thomas M.G., Warner B.W, & Randolph G.J. (2021). Enterically derived high-density lipoprotein restrains liver injury via the portal vein. Science (New York, N.Y.), 373(6553), eabe6729.

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Dual IHC Detection of Bri3 and Aβ

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Immunohistochemical staining for detection of Bri3 and Aβ were performed on 5 μm thin sections from formalin fixed paraffin embedded tissue. Brain sections were de-paraffinized in xylene and rehydrated through graded alcohol series from 99% to 70%. Sections were autoclaved in a Decloaking Chamber (Biocare Medical) in DIVA decloaker solution (Biocare Medical, Concord, USA) for 30 min. Sections were washed with Tris-buffered saline containing 0.05% tween 20 (TBST), and incubated first with peroxidase blocking solution (Dako) for 5 min, washed in TBST and then incubated for 10 min with Background punisher (Dako). Double stained slides were incubated for 45 min at RT with an antibody cocktail containing: mouse anti-AβPP/Aβ (6E10, 1:1000) and rabbit anti-Bri3 BRICHOS (1:500) diluted in Dako antibody diluent (Dako). Negative control slides were incubated with the Dako antibody diluent only. Slides were washed in TBST and incubated for 30 min at RT with Mach 2 Double Stain1 containing conjugated secondary anti-mouse HRP and anti-rabbit-AP antibody (Biocare Medical). Horseradish peroxidase staining was visualized with the permanent green (Biosite) and AP staining was visualized with permanent red (Biosite) solutions. Slides were counterstained in hematoxylin, dehydrated through graded alcohols, cleared in xylene and then mounted with Permount. Images were acquired at 40X magnification.

Dolfe L., Tambaro S., Tigro H., Del Campo M., Hoozemans J.J., Wiehager B., Graff C., Winblad B., Ankarcrona M., Kaldmäe M., Teunissen C.E., Rönnbäck A., Johansson J, & Presto J. (2018). The Bri2 and Bri3 BRICHOS Domains Interact Differently with Aβ42 and Alzheimer Amyloid Plaques. Journal of Alzheimer's Disease Reports, 2(1), 27-39.

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SARS-CoV-2 Lung Histopathology and Immune Response

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For each group analyzed (N = 5 mice/group), the right lung lobe was extracted, fixed in formalin and paraffin embedded as described^{29 (link)}. Lung sections were stained with Carstairs staining for histological analysis. Prior to conduct immunofluorescence assay, lung sections were deparaffined, hydrated then heat-induced epitope retrieval 16 h at 60 °C with Diva Decloaker solution (Biocare Medical, Pacheco, CA, USA). Immunostainings were preformed to detect SARS-CoV-2 N antigen and leukocyte infiltration using 20 µg/mL rabbit anti-N (Rockland chemicals, Limerick, PA, USA)/anti-rabbit IgG Alexa 488 (Jackson Immuno Research lab, West Grove, PA, USA) and 10 µg/mL biotinylated anti-CD45 antibodies (BD Bioscience, Franklin Lakes, NJ, USA)/anti Rat IgG Alexa Plus 647 (Thermo Fisher Scientific, Waltham, MA, USA). Slide were imaged using Axioscan 7 instrument (Carl Zeiss Microscopy, New York, USA) then black and white adjustment were performed with Zen lite 3.7 (Carl Zeiss Microscopy). Quantification of positive area signal for N and CD45 staining were performed using Fiji (ImageJ) threshold analyse tools.

Willett J.D., Gravel A., Dubuc I., Gudimard L., dos Santos Pereira Andrade A.C., Lacasse É., Fortin P., Liu J.L., Cervantes J.A., Galvez J.H., Djambazian H.H., Zwaig M., Roy A.M., Lee S., Chen S.H., Ragoussis J, & Flamand L. (2024). SARS-CoV-2 rapidly evolves lineage-specific phenotypic differences when passaged repeatedly in immune-naïve mice. Communications Biology, 7, 191.

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