Epitope retrieval solution 1

Manufactured by Leica

Sourced in Germany

Epitope Retrieval Solution 1 is a laboratory reagent designed to facilitate the exposure of antigenic sites, or epitopes, on tissue samples during immunohistochemical staining procedures. It is a critical component in the process of preparing samples for analysis.

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Lab products found in correlation

Bond max, by Leica (5 mentions) Bond rx, by Leica (3 mentions) Background reducing antibody diluent, by Agilent Technologies (3 mentions) Immuno wash, by StatLab (3 mentions) Peroxide block, by Leica (3 mentions) Polymer define detection system, by Leica (3 mentions) Bond dewaxing solution, by Leica (3 mentions) Anti rabbit hrp polymer, by Leica (3 mentions) Gtx135357, by GeneTex (3 mentions) Bond polymer refine detection system, by Leica (2 mentions) Bond polymer refine detection kit, by Leica (2 mentions) Bond dewax solution, by Leica (2 mentions) Glycerol solution, by Merck Group (2 mentions) Bond rxautomated ihc ish stainer, by Leica (2 mentions) Hoechst 33342, by LI COR (2 mentions) Anti pd l1 antibody clone sp142, by Roche (1 mentions) Mab4383, by Merck Group (1 mentions) Mouse igg1, by Cell Signaling Technology (1 mentions) Anti sdma, by Cell Signaling Technology (1 mentions) Epitope retrieval solution 2, by Leica (1 mentions)

Spelling variants of this product

Bond Epitope Retrieval Solution 1 (58 citations) Epitope Retrieval Solution (18 citations) Leica Epitope Retrieval 1 (4 citations) Bond Epitope Retrieval Solution 1 (ER1) (3 citations) BOND Novocastra Epitope Retrieval Solution 1 (2 citations)

12 protocols using epitope retrieval solution 1

PD-L1 Expression in FFPE Tumor Tissue

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An immunohistochemical study was performed on 5-µm-thick sections of FFPE tumor tissue using a fully automated immunohistochemistry and in situ hybridization machine (BOND-MAX; Leica, Wetzlar, Germany). The primary antibody used was a monoclonal rabbit anti-PD-L1 antibody (clone SP142; Roche/Ventana, Tucson, AZ, USA) at a dilution of 1:100. Heat-induced epitope retrieval was performed using a citrate-based buffer (Epitope Retrieval Solution 1; Leica, Wetzlar, Germany) at 100 °C for 20 min. Under a microscope, cells with definite membranous staining were considered positive. TCs can be easily distinguished from ICs by their much larger nuclear size (Figure 6c–f). The percentages of TCs and ICs positive for PD-L1 were evaluated separately. The evaluation was first performed independently by two senior pathologists (W.-Y. Chuang and C. Hsueh) without knowing the clinical data. For discrepant results, a consensus percentage was determined by examination under a dual head microscope. For both TCs and ICs, low PD-L1 expression was defined as a percentage of positive cells lower than the median of all cases (otherwise regarded as high expression).

Liu Y.J., Tsang N.M., Hsueh C., Yeh C.J., Ueng S.H., Wang T.H, & Chuang W.Y. (2018). Low PD-L1 Expression Strongly Correlates with Local Recurrence in Epstein-Barr Virus-Positive Nasopharyngeal Carcinoma after Radiation-Based Therapy. Cancers, 10(10), 374.

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SARS-CoV-2 Antigen Immunohistochemistry

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Staining for SARS-CoV-2 antigen was achieved on the Bond RX automated system with the Polymer Define Detection System (Leica) used per manufacturer’s protocol. Tissue sections were dewaxed with Bond Dewaxing Solution (Leica) at 72°C for 30 min then subsequently rehydrated with graded alcohol washes and 1x Immuno Wash (StatLab). Heat-induced epitope retrieval (HIER) was performed using Epitope Retrieval Solution 1 (Leica), heated to 100°C for 20 minutes. A peroxide block (Leica) was applied for 5 min to quench endogenous peroxidase activity prior to applying the SARS-CoV-2 antibody (1:2000, GeneTex, GTX135357). Antibodies were diluted in Background Reducing Antibody Diluent (Agilent). The tissue was subsequently incubated with an anti-rabbit HRP polymer (Leica) and colorized with 3,3’-Diaminobenzidine (DAB) chromogen for 10 min. Slides were counterstained with hematoxylin.

Li D., Martinez D.R., Schäfer A., Chen H., Barr M., Sutherland L.L., Lee E., Parks R., Mielke D., Edwards W., Newman A., Bock K.W., Minai M., Nagata B.M., Gagne M., Douek D.C., DeMarco C.T., Denny T.N., Oguin TH I.I.I., Brown A., Rountree W., Wang Y., Mansouri K., Edwards R.J., Ferrari G., Sempowski G.D., Eaton A., Tang J., Cain D.W., Santra S., Pardi N., Weissman D., Tomai M.A., Fox C.B., Moore I.N., Andersen H., Lewis M.G., Golding H., Seder R., Khurana S., Baric R.S., Montefiori D.C., Saunders K.O, & Haynes B.F. (2022). Breadth of SARS-CoV-2 Neutralization and Protection Induced by a Nanoparticle Vaccine. bioRxiv.

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Immunohistochemical Analysis of Mouse Brain

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Upon sacrifice, mouse brains were formalin fixed and paraffin embedded. Tissue sections were processed for immunohistochemical staining with the BOND-MAX autostainer (Leica, Germany). Antigen retrieval was performed at 100 °C for 20 min using Epitope Retrieval Solution 1 (AR9961, Leica). Primary antibody or IgG control incubation was for 1 h and signal detected using the BOND™ Polymer Refine Detection System (DS9800, Leica) according to manufacturer’s instructions. Primary antibodies were anti-SDMA (Cell Signalling Technologies, #13,222, 1:200), anti-H4R3me2s (EpiGentek, a-3718, 1:150), anti-human nuclear antigen (Sigma-Aldrich, MAB4383, 1:200). Isotype controls were mouse IgG (1:150, ReliaTech IgG-010) or mouse IgG1 (1:500, Cell Signalling Technology #5415). Images were analysed using FIJI/ImageJ (v1.53c), and the Trainable Weka Segmentation plugin (v3.2.34)^{101 (link)} was used for segmentation of the images.

Brown E.J., Balaguer-Lluna L., Cribbs A.P., Philpott M., Campo L., Browne M., Wong J.F., Oppermann U., Carcaboso Á.M., Bullock A.N, & Farnie G. (2024). PRMT5 inhibition shows in vitro efficacy against H3K27M-altered diffuse midline glioma, but does not extend survival in vivo. Scientific Reports, 14, 328.

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Immunohistochemical Detection of SARS-CoV-2

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Staining for SARS-CoV-2 antigen was achieved on the Bond RX automated system with the Polymer Define Detection System (Leica) used per manufacturer’s protocol. Tissue sections were dewaxed with Bond Dewaxing Solution (Leica) at 72 °C for 30 min then subsequently rehydrated with graded alcohol washes and 1× Immuno Wash (StatLab). Heat-induced epitope retrieval (HIER) was performed using Epitope Retrieval Solution 1 (Leica), heated to 100 °C for 20 min. A peroxide block (Leica) was applied for 5 min to quench endogenous peroxidase activity prior to applying the SARS-CoV-2 antibody (1:2000, GeneTex, GTX135357). Antibodies were diluted in Background Reducing Antibody Diluent (Agilent). The tissue was subsequently incubated with an anti-rabbit HRP polymer (Leica) and colorized with 3,3’-Diaminobenzidine (DAB) chromogen for 10 min. Slides were counterstained with hematoxylin.

Li D., Martinez D.R., Schäfer A., Chen H., Barr M., Sutherland L.L., Lee E., Parks R., Mielke D., Edwards W., Newman A., Bock K.W., Minai M., Nagata B.M., Gagne M., Douek D.C., DeMarco C.T., Denny T.N., Oguin TH I.I.I., Brown A., Rountree W., Wang Y., Mansouri K., Edwards R.J., Ferrari G., Sempowski G.D., Eaton A., Tang J., Cain D.W., Santra S., Pardi N., Weissman D., Tomai M.A., Fox C.B., Moore I.N., Andersen H., Lewis M.G., Golding H., Seder R., Khurana S., Baric R.S., Montefiori D.C., Saunders K.O, & Haynes B.F. (2022). Breadth of SARS-CoV-2 neutralization and protection induced by a nanoparticle vaccine. Nature Communications, 13, 6309.

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Immunohistochemical Evaluation of PDCD4 Expression

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Immunohistochemistry was performed on 4-μm to 5-μm thick FFPE sections from each tissue sample. Staining was automatically performed using a fully automated system (Bond™-maX; Leica, Newcastle Upon Tyne, UK). Sections were pre-treated using heat-mediated antigen retrieval with sodium citrate buffer (pH6, Epitope Retrieval Solution 1, Leica) for 30 minutes at 99°C. Specimens were then incubated with rabbit polyclonal anti-PDCD4 (catalog No. HPA001032; Atlas Antibodies, Stockholm, Sweden; 1:200) and detected using the Bond Polymer Refine Detection Kit (Leica) according to the manufacturer’s protocols. The staining was visualized with 3,3’-diaminobenzidine (DAB) and the slides were lightly counterstained with hematoxylin. Sections were then dehydrated, cleared, and mounted. We used samples from FFPE human tonsil tissue as positive controls and serum without the primary antibody as negative control. PDCD4 nuclear expression was jointly scored by 2 pathologists (LN and RC) in a whole section for each case, unaware of any clinical information. A semi-quantitatively 4-tiered scale based on the percentage of positive cells was used, with 0 indicating no stain, 1 indicating 1% to 30% staining, 2 indicating 31% to 70% staining, and 3 indicating 71% to 100% staining.

Nicolè L., Cappellesso R., Sanavia T., Guzzardo V, & Fassina A. (2018). MiR-21 over-expression and Programmed Cell Death 4 down-regulation features malignant pleural mesothelioma. Oncotarget, 9(25), 17300-17308.

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SARS-CoV-2 Nucleocapsid Antigen Detection

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Staining for SARS-CoV-2 nucleocapsid antigen was performed by the Bond RX automated system with the Polymer Define Detection System (Leica) following the manufacturer’s protocol. Tissue sections were dewaxed with Bond Dewaxing Solution (Leica) at 72°C for 30 min, then subsequently rehydrated with graded alcohol washes and 1x Immuno Wash (StatLab). Heat-induced epitope retrieval (HIER) was performed using Epitope Retrieval Solution 1 (Leica) and by heating the tissue section to 100°C for 20 min. A peroxide block (Leica) was applied for 5 min to quench endogenous peroxidase activity prior to applying the SARS-CoV-2 nucleocapsid antibody (1:2000, GeneTex, GTX135357). Antibodies were diluted in Background Reducing Antibody Diluent (Agilent). The tissue was subsequently incubated with an anti-rabbit HRP polymer (Leica) and colorized with 3,3’-Diaminobenzidine (DAB) chromogen for 10 min. Slides were counterstained with hematoxylin.

Saunders K.O., Lee E., Parks R., Martinez D.R., Li D., Chen H., Edwards R.J., Gobeil S., Barr M., Mansouri K., Alam S.M., Sutherland L.L., Cai F., Sanzone A.M., Berry M., Manne K., Bock K.W., Minai M., Nagata B.M., Kapingidza A.B., Azoitei M., Tse L.V., Scobey T.D., Spreng R.L., Wes Rountree R., DeMarco C.T., Denny T.N., Woods C.W., Petzold E.W., Tang J., Oguin TH I.I.I., Sempowski G.D., Gagne M., Douek D.C., Tomai M.A., Fox C.B., Seder R., Wiehe K., Weissman D., Pardi N., Golding H., Khurana S., Acharya P., Andersen H., Lewis M.G., Moore I.N., Montefiori D.C., Baric R.S, & Haynes B.F. (2021). Neutralizing antibody vaccine for pandemic and pre-emergent coronaviruses. Nature, 594(7864), 553-559.

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Immunohistochemical Analysis of Cellular Markers

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Immunohistochemical studies were conducted on formalin-fixed, paraffin-embedded sections 4 to 5 μm thick obtained from each tissue sample. Staining was done automatically using a fully automated system (Bond™-maX; Leica, Newcastle Upon Tyne, UK). Sections were pretreated using heat-mediated antigen retrieval with sodium citrate buffer (pH6, Epitope Retrieval Solution 1, Leica) for 30 minutes at 99°C. Specimens were then incubated with the panel of primary antibodies listed in Table 2, according to the manufacturer ' s instructions. Sections were then lightly counterstained with hematoxylin. Appropriate positive and negative controls were run concurrently. Immunoreactions for β-arrestin-1 were detectable in the cytoplasm, those for E-cadherin and Zeb1/Zeb2 in the cellular membrane and nucleus, respectively. Immunohistochemical reactions were scored semiquantitatively by two pathologists (R.C. and L.N.) on a two-tiered scale, based on the percentage of positive tumor cells, as: negative (0-30%); or positive (>30%).

Marioni G., Nicolè L., Cappellesso R., Marchese-Ragona R., Fasanaro E., Di Carlo R., La Torre F.B., Nardello E., Sanavia T., Ottaviano G, & Fassina A. (2019). β-Arrestin-1 expression and epithelial-to-mesenchymal transition in laryngeal carcinoma. The International journal of biological markers, 34(1).

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Immunohistochemical Profile of Cytokeratins

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Serial sections at 3.5 μm were cut from Formalin Fixed Paraffin Embedded (FFPE) tissue blocks, mounted onto adhesive slides (Surgipath Snowcoat) and dried at 65ºC for 15 minutes. Each series of sections were stained on Bond III Automated Immunohistochemistry System Leica Stainer using Leica Bond Polymer Refine detection with a DAB chromogen (0.5% Copper Sulphate in PBS buffer). All antigen retrieval methods were performed on-board, following Queen Alexandra Hospital laboratory protocols, using antigen-unmasking solutions: Epitope Retrieval Solution 1 (ER1) at pH 6.0 (Leica, AR9961) and Epitope Retrieval Solution 2 (ER2) at pH 9.0 (Leica, AR9640) for 20 minutes. Each run included a positive control (normal endometrium) and a negative control where the primary antibody was omitted.
Cytokeratin markers tested were, as follows: AE1/3, CAM5.2, HMWCK, MNF116, CK5, CK6, CK7, CK8/18, CK14, CK17, CK19 and CK20. Additionally, staining for ER, PR, CD10 and Cyclin D1 was performed. Details of antibodies (sources, clones, working dilutions and antigen retrieval pre-treatment) are summarised in Table 2.
The staining was assessed with a semi-quantitative method as shown in Table 3.

Rahimi S., Akaev I., Marani C., Chopra M, & Yeoh C.C. (2019). Immunohistochemical Expression of Different Subtypes of Cytokeratins by Endometrial Stromal Sarcoma. Applied immunohistochemistry & molecular morphology : AIMM, 27(6).

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FFPE Tissue IHC Staining Protocol

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FFPE tissue sections (4 μm) were subjected to heat-mediated antigen retrieval with Epitope Retrieval 1 solution (pH6, Leica Biosystems) for 20 minutes, then stained with the Bond RX system (Leica Biosystems) using the standard protocol. The sections were incubated with anti-CD8 at 1:25 dilution (Thermo Scientific #MS-457) or anti-PD-L1 at 1:100 dilution (Cell Signaling Technology #13684S). Visualization was performed using the Bond Polymer Refine Detection Kit (Leica Biosystems) according to the manufacturer’s instructions.

Haymaker C.L., Kim D., Uemura M., Vence L.M., Phillip A., McQuail N., Brown P.D., Fernandez I., Hudgens C.W., Creasy C., Hwu W.J., Sharma P., Tetzlaff M.T., Allison J.P., Hwu P., Bernatchez C, & Diab A. (2017). Metastatic Melanoma Patient Had a Complete Response with Clonal Expansion after Whole Brain Radiation and PD-1 Blockade. Cancer immunology research, 5(2), 100-105.

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Multimodal Tissue Imaging by t-CyCIF

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The t-CyCIF experimental protocol was conducted as previously
described (Du et al., 2019 (link); Lin et al., 2018 ). In brief, the mouse
and human lung adenocarcinoma and human melanoma FFPE slides were baked at
60°C for 30 min, dewaxed using Bond Dewax Solution (Leica Biosystems)
at 72°C, and antigen retrieval was performed with Epitope Retrieval 1
Solution (Leica Biosystems) at 100°C for 20 minutes using the BOND RX
Automated IHC/ISH Stainer (Leica Biosystems). All antibodies were diluted in
Odyssey Intercept Buffer (plus Hoechst 33342 0.25 μg/mL; LI-COR
Biosciences) and incubated overnight at 4°C in the dark. See the
Key Resources Table for the
complete list of antibodies. Slides were coverslipped using 20–50%
glycerol solution (Sigma-Aldrich) in PBS. Images were taken using DAPI,
FITC, Cy3, and Cy5 channels on the RareCyte CyteFinder Instrument
(20x/0.75NA objective lens). After imaging, the fluorophores were
inactivated with photobleaching solution (4.5% H2O2 and 20 mM NaOH in PBS)
for 45 minutes under LED ligfFigurehts.

Burger M.L., Cruz A.M., Crossland G.E., Gaglia G., Ritch C.C., Blatt S.E., Bhutkar A., Canner D., Kienka T., Tavana S.Z., Barandiaran A.L., Garmilla A., Schenkel J.M., Hillman M., de los Rios Kobara I., Li A., Jaeger A.M., Hwang W.L., Westcott P.M., Manos M.P., Holovatska M.M., Hodi F.S., Regev A., Santagata S, & Jacks T. (2021). Antigen Dominance Hierarchies Shape TCF1+ Progenitor CD8 T Cell Phenotypes in Tumors. Cell, 184(19), 4996-5014.e26.

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