Dab enhancer

Manufactured by Leica

Sourced in Germany

The DAB enhancer is a laboratory equipment product designed to enhance the visibility of DAB (3,3'-Diaminobenzidine) staining in immunohistochemistry and other related applications. It is a specialized reagent that can amplify the signal intensity of DAB-based detection systems, improving the overall sensitivity and clarity of the staining results.

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Bond Dab Enhancer (7 citations)

11 protocols using dab enhancer

IHC Protocol for ZMIZ1 Detection in Breast Cancer

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Antibody specificity was confirmed on formalin-fixed cell pellets of MCF7 cells (Supplementary Fig. 1, see section on supplementary materials given at the end of this article). Patient tissue samples were run on Leica’s Polymer Refine Detection System (DS9800) using their standard template on the automated Bond-III platform. MCF7 cells were cultured in estrogen-rich complete DMEM media with 10% BS and glutamine. Post-siRNA knockdown of ZMIZ1 were used as negative control to ensure antibody specific. Dewaxing and re-hydration prior to IHC were automated on the Leica ST5020, along with the post-IHC dehydration and clearing. Sections were mounted using Leica’s coverslipper CV5030. The specific antibody targeting ZMIZ1 was purchased from R&D Systems (AF8107) and used at a concentration of 2 µg/mL (1:250 dilution). The sodium citrate pre-treatment was run at 100˚C. The secondary (post-primary) was rabbit anti-sheep from Jackson ImmunoResearch (r313-005-003), diluted 1:500. DAB Enhancer was included as an ancillary reagent (Leica, AR9432).
Patient tissue samples were processed as described for the MCF7 fixed cell pellets. ER-α antibody was purchased from Novacastra (NCL-ER-6F11/2) and samples processed as previously described (Bruna et al. 2016 (link)).

Zhao W., Rose S.F., Blake R., Godicelj A., Cullen A.E., Stenning J., Beevors L., Gehrung M., Kumar S., Kishore K., Sawle A., Eldridge M., Giorgi F.M., Bridge K.S., Markowetz F, & Holding A.N. (2024). ZMIZ1 enhances ERα-dependent expression of E2F2 in breast cancer. Journal of Molecular Endocrinology, 73(1), e230133.

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Standardized Ki-67 Immunohistochemistry Protocol

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Ki-67 scores were retrospectively obtained from pathology reports at initial diagnosis on the primary tumor prior to any treatment. IHC staining on formalin-fixed paraffin-embedded tissue sections was performed. Mind Bomb 1 mouse monoclonal antibody (manufactured by Dako) was used to detect Ki-67. The staining protocol included de-paraffinization (30 minutes at 72°C) and rehydration with antigen retrieval performed at 100°C for 20 minutes with Tris-EDTA buffer, pH 6.0. Endogenous peroxidase was blocked with 3% peroxide for 5 minutes. Primary anti-Ki-67 antibody (Dako, clone MIB-1) was applied at 1:100 dilution for 15 minutes. Post primary antibody detection was carried out using a commercial polymer system (Bond Polymer Refine Detection, Leica), and stain development was achieved by incubation with diaminobenzidine (DAB) and DAB enhancer (Leica). The robust quality control and quality improvement program of the IHC lab at MDA was fully applied to the anti-Ki-67 IHC assay. A positive control was added to every IHC run (reference tonsil tissue, batch control) and was reviewed by a member of the IHC medical directorship team. Records of batch control results were documented daily in internal laboratory records. Stains were evaluated by designated breast pathologists, who visually estimated the percentage of positively staining invasive carcinoma cells.

Ning J., Fouad T.M., Lin H., Sahin A.A., Lucci A., Woodward W.A., Krishnamurthy S., Tripathy D., Ueno N.T, & Shen Y. (2019). The impact of Ki-67 in the context of multidisciplinary care in primary inflammatory breast cancer. Journal of Cancer, 10(12), 2635-2642.

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Immunohistochemical Analysis of Intestinal Tissue

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The small intestine and colon were opened and fixed for 24 h in 4% PFA. The tissue was paraffin embedded and sectioned. RFP and β-catenin immunohistochemistry were carried out using a Bond Max autostainer (Leica), with sodium citrate, pH 6.0 (10 mM) antigen retrieval. Slides were blocked with 3% hydrogen peroxide, followed by incubation using an Avidin/Biotin Blocking Kit (Vector Laboratories). Anti-RFP (1:100, Abcam ab34771) and β-catenin (BD biosciences, 610154, 0.25 μg/mL) primary antibodies were used. For β-catenin IHC, a mouse-on-mouse blocking step was added (Vector Laboratories, MKB-2213). Secondary antibodies used were biotinylated donkey and biotinylated donkey anti-rabbit (1:250, Jackson ImmunoResearch, 711-065-152) and biotinylated rabbit anti-mouse IgG1 (1:500, Abcam, ab125913). Slides were incubated with streptavidin coupled with horseradish peroxidase (HRP), and colour developed using diaminobenzidine (DAB) and DAB Enhancer (Leica).

Thorsen A.S., Khamis D., Kemp R., Colombé M., Lourenço F.C., Morrissey E, & Winton D. (2021). Heterogeneity in clone dynamics within and adjacent to intestinal tumours identified by Dre-mediated lineage tracing. Disease Models & Mechanisms, 14(1), dmm046706.

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Immunohistochemical Analysis of CD3+ T Cells

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Tissues were fixed in 10% neutral buffered formaldehyde. Immunohistochemistry was performed for CD3 using a Leica Bond III immunostainer. Deparaffinization and rehydration were conducted before antigen retrieval was performed using Tris EDTA (pH 9) (ER2) for 20 min at 100°C, incubation in a rabbit polyclonal anti-CD3 antibody (A0452) for 15 min, HRP-linked anti-rabbit polymer for 8 min, diaminobenzidine for 10 min, and DAB Enhancer (Leica) for 10 min. Counterstaining was with hematoxylin for 2 min. The slides were scanned at 20× on a Leica AT2 and subsequently analyzed in a blinded manner using the Cytonuclear v1.4 algorithm on the HALO platform.

Flint T.R., Janowitz T., Connell C.M., Roberts E.W., Denton A.E., Coll A.P., Jodrell D.I, & Fearon D.T. (2016). Tumor-Induced IL-6 Reprograms Host Metabolism to Suppress Anti-tumor Immunity. Cell Metabolism, 24(5), 672-684.

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Immunohistochemical Staining of Iron Metabolism Proteins

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Stainings were performed using the Bond Polymer Refine Detection system (Leica Biosystems, Newcastle-Upon-Tyne, UK) and provided materials on a Leica Bond Max and Autostainer XL apparatus for DMT1 (Novus Biologicals, Abingdon, UK, H00004891-M01, 1:2000), ZIP8 (Protein Tech, Manchester, UK, 20459-1-AP, 1:500), ZIP14 (Atlas Antibodies, Bromma, Sweden, HPA016508, 1:1000), L-ferritin (Abcam, Cambridge, UK, ab69090, 1:2000), H-ferritin (Abcam ab65080, 1:4000), ferroportin (Abcam ab85370, 1:300) and HO-1 (Abcam ab13243, 1:100). After antigen retrieval (Bond ER1 or ER2), sections were incubated with primary antibody for 15 min diluted in Bond Primary Antibody diluent or Antibody diluent with Background Reducing Components (Dako Agilent, Stockport, UK) and secondary antibody for 8 min. Signal visualization was performed with Polymer Refine for 8 min, DAB for 10 min and DAB enhancer (Leica Biosystems). Afterwards, nuclei were counterstained with haematoxylin and images were taken with a Leica DM 2000 microscope connected to a Leica Microsystem Ltd camera, NanoZoomer whole slide imager (Hamamatsu Photonics, Welwyn Garden City, UK) or a VisionTek digital microscope (Sakura Finetek, Alphen aan den Rijn, NL). Appropriate negative control stainings were included for all primary and secondary antibodies (Supplementary Figure 1 and 2, respectively).

van Raaij S., van Swelm R., Bouman K., Cliteur M., van den Heuvel M.C., Pertijs J., Patel D., Bass P., van Goor H., Unwin R., Srai S.K, & Swinkels D. (2018). Tubular iron deposition and iron handling proteins in human healthy kidney and chronic kidney disease. Scientific Reports, 8, 9353.

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Immunohistochemical Profiling of RA Synovial Tissue

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Formalin-fixed paraffin-embedded (FFPE) RA synovial tissue sections were stained with haematoxylin and eosin (H&E). For IHC, sections were incubated with antibodies specific for B cells (CD20, [Dako L26]; 1:20), T cells (CD3 [Cell Marque MRQ-39]; 1:50), macrophages (CD68 (Leica KP1); 1:100) and endothelial cells (vWF (Dako A0082); 1:600) diluted in Primary Antibody Diluent BOND (Leica Biosystems), using automated IHC following heat-mediated epitope retrieval and diaminobenzidine chromogen (DAB; Leica) or Bond Polymer Refine Red (Leica Biosystems) detection. For p53β, IHC was done manually following antigen retrieval (heat-mediated Tris–EDTA, pH 9.0) using rabbit polyclonal antibody ‘79.3’ (1:150) in van Gogh diluent (Biocare Medical) overnight at 4 °C, detection by EnVision Dual Link (Dako) followed by DAB (Dako) with DAB enhancer (Leica Biosystems).

Wiles A.K., Mehta S., Millier M., Woolley A.G., Li K., Parker K., Kazantseva M., Wilson M., Young K., Bowie S., Ray S., Slatter T.L., Stamp L.K., Hessian P.A, & Braithwaite A.W. (2023). Activated CD90/Thy-1 fibroblasts co-express the Δ133p53β isoform and are associated with highly inflamed rheumatoid arthritis. Arthritis Research & Therapy, 25, 62.

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RNAscope Assay for TP53 Isoforms

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A custom probe to the unique region of ∆133TP53 and TP53β was made by Advanced Cell Diagnostics (Advanced Cell Diagnostics, Newark, CA, USA). The probe was designed to Δ133TP53β reference sequence DQ186651.1 with the probes between nucleotides +97 and +277 unique to Δ133TP53 isoforms, but excluding the upstream AluJb repeat^{47 (link)}. To increase the amount of sequence available for probe stability, nucleotides +847–1001 were also included. Probes to VEGFA were used on human (reference number 423161) and mouse cells (reference 436961, Advanced Cell Diagnostics, Newark, CA, USA).
Formalin fixed paraffin-embedded cell clots and tumors were cut into 5 µm sections. The RNAscope method used the manual assay 2.5 protocol with Protease Plus reagent for protein digestion and the 2.5HD reagent kit brown for detection of the probe according to the manufacturer’s instructions. The assay was optimized using paraffin-embedded cell clots containing MCF7 and TP53 null Saos-2 cells (Supplementary Fig. S2). Following addition of DAB, DAB enhancer was added (Leica Biosystems, Wetzlar Germany). Positive cells were identified using the Aperio Scancope CS digital pathology system and quantified using the Aperio RNA ISH Algorithm (Aperio, Vista, California, USA). Slides were evaluated by two blinded examiners.

Kazantseva M., Mehta S., Eiholzer R.A., Gimenez G., Bowie S., Campbell H., Reily-Bell A.L., Roth I., Ray S., Drummond C.J., Reid G., Joruiz S.M., Wiles A., Morrin H.R., Reader K.L., Hung N.A., Baird M.A., Slatter T.L, & Braithwaite A.W. (2019). The Δ133p53β isoform promotes an immunosuppressive environment leading to aggressive prostate cancer. Cell Death & Disease, 10(9), 631.

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Immunohistochemical Staining of p53β and CA9

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For immunohistochemical (IHC) staining, 4‐μm sections from paraffin‐embedded tissues were used. The KJC8 antibody towards p53β was generously provided by the Bourdon Laboratory (University of Dundee, Ninewells Hospital and Medical School, Dundee, UK) and was detected using EnVision Dual Link (Dako, Glostrup, Denmark) and diaminobenzidine chromogen (DAB), with DAB enhancer (Leica Biosystems, Wetzlar, Germany). The CA9 antibody (MRQ‐54; Cell Marque, Rocklin, CA, USA) was used to detect CA9‐positive cells using an automated IHC method (BOND RX automated stainer; Leica Biosystems, Wetzlar, Germany).

Kazantseva M., Eiholzer R.A., Mehta S., Taha A., Bowie S., Roth I., Zhou J., Joruiz S.M., Royds J.A., Hung N.A., Slatter T.L, & Braithwaite A.W. (2018). Elevation of the TP53 isoform Δ133p53β in glioblastomas: an alternative to mutant p53 in promoting tumor development. The Journal of Pathology, 246(1), 77-88.

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Quantifying TP53 Isoforms in Tumors

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Probes included a custom probe to the unique region of ∆133TP53 and TP53β made by Advanced Cell Diagnostics named ∆133TP53 and described elsewhere [2 (link),3 (link)], as well as the probes ubiquitin C (UBC, positive control) and DapB (negative control, Advanced Cell Diagnostics, Newark, CA, USA). Formalin-fixed paraffin-embedded tumors or cell clots were cut into 5 µm sections. The RNAscope method used the manual assay method with Protease Plus reagent for protein digestion and the 2.5HD reagent kit brown according to the manufacturer’s instructions. Following the addition of DAB to the ∆133TP53 and DapB assays, DAB enhancer was added (Leica Biosystems, Wetzlar, Germany). Positive cells and the number of dots per positive cell were identified using the Aperio Scancope CS digital pathology system and quantified using the Aperio RNA ISH Algorithm (Aperio, Vista, CA, USA). The slides were evaluated by two blinded examiners. The percentage of positive cells out of the total cell number was measured. Ten fields (×400 magnification) were chosen at random and the percentage of positive cells out of the total cells counted. Tumors with no positive cells in the 10 random fields were examined over the entire tissue section for the presence of positive cells.

Jesus A.N., Taha A., Wang D., Mehta P.M., Mehta S., Reily-Bell A., Lekamlage S.P., Saraiva A.M., Tahmeedzaman T., Ziad F., Thotathil Z., Gan P.Y., Royds J., Braithwaite A., Hung N, & Slatter T.L. (2023). Increased Expression of the Δ133p53β Isoform Enhances Brain Metastasis. International Journal of Molecular Sciences, 24(2), 1267.

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Histological Analysis of Testes Tissue

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Testes were fixed in neutral buffered formalin (NBF) for 24 h, transferred to 70% ethanol, machine processed and paraffin embedded. Formalin-fixed paraffin-embedded (FFPE) sections of 3 µm thickness were used for all histological stains and immunohistochemistry (IHC).
For Periodic Acid Schiff (PAS) stainings slides were dewaxed, washed in water and placed in 0.5% Periodic Acid (Sigma P0430) for 5 min. After three washes in ultra-pure water, slides were placed in Schiff reagent (Thermo Fisher Scientific, J/7300/PB08) for 15–30 min in a closed container and washed again three times in ultra-pure water. Counterstain was performed using Mayers Haematoxylin (Thermo Fisher Scientific, LAMB/170-D) for 40 s followed by rinsing in tap water, dehydration and mounting.
IHC was performed on FFPE sections using the Bond™ Polymer Refine Kit (DS9800, Leica Microsystems) on the automated Bond Platform. Anti-phospho-Histone H3 (Ser10) (pH3) antibody (Upstate, 06-570, 1:200 dilution) was used with DAB Enhancer (Leica Microsystems, AR9432) and heat-induced epitope retrieval was performed for 10 min at 100 °C on the Bond platform with sodium citrate. All slides were scanned using Aperio XT (Leica Biosystems) and PH3 intensities were quantified using the Aperio eSlide Manager (Leica Biosystems).

Ernst C., Eling N., Martinez-Jimenez C.P., Marioni J.C, & Odom D.T. (2019). Staged developmental mapping and X chromosome transcriptional dynamics during mouse spermatogenesis. Nature Communications, 10, 1251.

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