Polymer refine detection reagents

Manufactured by Leica

Sourced in United States

Polymer Refine Detection reagents are a set of laboratory chemicals used in the process of immunohistochemistry. These reagents are designed to enhance the visualization and detection of target proteins or antigens in tissue samples. The core function of these reagents is to amplify the signal generated during the immunohistochemical staining process, thereby improving the sensitivity and clarity of the final results.

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Lab products found in correlation

Bond max immunostainer, by Leica (5 mentions) Ls b4861, by LifeSpan BioSciences (2 mentions) Antibody diluent, by Leica (2 mentions) Phospho akt ser473, by GeneTex (2 mentions) Phospho mtor ser2448, by Cell Signaling Technology (2 mentions) Edta ph 9, by Leica (1 mentions) Insulin, by Cell Signaling Technology (1 mentions) Bond max, by Leica (1 mentions) Phospho s6 ser235 236, by Cell Signaling Technology (1 mentions) F4 80, by Cell Signaling Technology (1 mentions) Ab32500, by Abcam (1 mentions) Sodium citrate, by Leica (1 mentions) Cytokeratin 17 19, by Cell Signaling Technology (1 mentions) Alcian blue, by StatLab (1 mentions) Phospho p70s6k thr389, by Cell Signaling Technology (1 mentions) Rotary microtome, by Leica (1 mentions) Hematoxylin and eosin reagents, by Leica (1 mentions)

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Bond Polymer Refine Detection reagents (5 citations)

6 protocols using polymer refine detection reagents

Histological and Immunohistochemical Analyses

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Specimens were fixed in 10% neutral buffered formalin (Surgipath Leica, Buffalo Grove, IL) and paraffin embedded. Five-micrometer sections were cut with a rotary microtome (Leica). Histologic staining used SelecTech hematoxylin and eosin (H&E) reagents (Surgipath). Staining with Alcian Blue or Trichrome (both from American MasterTech, Lodi, CA) was performed as per manufacturers' instructions.
Antigen retrieval for immunohistochemistry was optimized with sodium citrate (pH 6.0) or EDTA (pH 9.0; Leica). Primary antibodies were against phospho-Akt Ser473 (GeneTex, Irvine, CA), phospho-mTor Ser2448 (Cell Signaling Technology, Danvers, MA), phospho-p70S6K Thr389 (Upstate Cell Signaling, Temecula, CA or LifeSpan BioSciences, Seattle, WA), and phospho-p70S6K Thr389 (Cell Signaling Technology), mucin-4 (Muc-4; LifeSpan BioSciences), α-2 smooth muscle actin (α-SMA; Novus, Littleton, CO), cytokeratin 17/19 (Cell Signaling Technology), and Ki67 (Abcam, Cambridge, MA). Detection used Polymer Refine Detection reagents (Leica). A Bond-Max Immunostainer and Polymer Refine Detection reagents (Leica) were used.

Albury T.M., Pandey V., Gitto S.B., Dominguez L., Spinel L.P., Talarchek J., Klein-Szanto A.J., Testa J.R, & Altomare D.A. (2015). Constitutively Active Akt1 Cooperates with KRasG12D to Accelerate In Vivo Pancreatic Tumor Onset and Progression. Neoplasia (New York, N.Y.), 17(2), 175-182.

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Immunohistochemical Analysis of CCT2 in Murine Breast Cancer

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Murine breast cancer tumors obtained through in vivo experiment described above. Samples were stained for CCT2 using anti-CCTβ antibody (LS-B4861; LifeSpan Biosciences) Antibodies were diluted 1:100 in Antibody Diluent (Leica). Staining of tissue arrays was performed using a Bond-Max Immunostainer (Leica), with an epitope retrieval buffer of EDTA pH 9.0 (Leica). Polymer Refine Detection reagents (Leica) were used, which include a hematoxylin counterstain. Scoring of staining was performed by a surgical pathologist as previously published^{14 (link),16 (link)}.

Showalter A.E., Martini A.C., Nierenberg D., Hosang K., Fahmi N.A., Gopalan P., Khaled A.S., Zhang W, & Khaled A.R. (2020). Investigating Chaperonin-Containing TCP-1 subunit 2 as an essential component of the chaperonin complex for tumorigenesis. Scientific Reports, 10, 798.

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Pancreatic Immunohistochemistry and Islet Morphometry

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Pancreatic tissue was fixed in 10% neutral buffered formalin (Surgipath Leica, Buffalo Grove, IL) and embedded in paraffin for sectioning and processing as previously described (Albury et al. 2015 (link)). Slides were stained for hematoxylin and eosin (Surgipath) or immunohistochemistry using the Polymer Refine Detection reagents (Leica) on the Bond-Max immunostainer (Leica). Antigen retrieval was optimized using sodium citrate (pH 6) or EDTA (pH 9) buffers (Leica). The following antibodies were used: phospho-Akt Ser473 (GeneTex, Irvine, CA), also phospho-mTor Ser2448, phospho-S6 Ser235/236, glucagon, and insulin (all from Cell Signaling Technology, Danvers, MA). All slides processed on the immunostainer were run with a negative control, which was treated with antibody diluent instead of the primary antibody, to ensure antibody specificity. Images were taken using a Leica DM 2000 microscope with 5X, 10X, or 40X objectives. Islet size was measured in the whole pancreas of three mice per genotype. The mice selected had no significant lesions (NSL) at the time of necropsy, as described by a pathologist. The pancreas was sectioned into 5μm sections and every 25^th section was H&E stained. A total of 50 islets from three sections were analyzed per mouse. Islet diameter was determined using measuring tools available on an Axio Imaging System (Zeiss, Oberkochen, Germany).

Albury-Warren T.M., Pandey V., Spinel L.P., Masternak M.M, & Altomare D.A. (2015). Prediabetes Linked to Excess Glucagon in Transgenic Mice with Pancreatic Active AKT1. The Journal of endocrinology, 228(1), 49-59.

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Quantifying Tumor-Associated Macrophages by IHC

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Tissues were fixed in 10% neutral buffered formalin (Surgipath Leica, Buffalo Grove, IL, USA), embedded in paraffin, sliced in 5 μm sections, and dried at 65 °C for 1 h. Slides were used for IHC staining using Polymer Refine Detection reagents (Leica) on a Bond-Max immunostainer (Leica, Buffalo Grove, IL, USA). Antigen retrieval for IHC was optimized with sodium citrate (pH 6.0) or EDTA (pH 9.0). Primary antibodies included F4/80 (Cell Signaling Technology, Danvers, MA, USA; 70076S), CD86 (Cell Signaling Technology, Danvers, MA, USA; 19589S) and Ym1 (STEMCELL Technologies, Vancouver, Canada; 60130). Stained sections were assessed by a pathologist and poorly differentiated tumor regions were chosen for quantification. IHC staining was quantified using the Keyence BZ-X800 analysis software. Where possible, serial sections were imaged, and samples from three mice per treatment group and two fields of view per histology specimen were used for quantification. Results are reported as mean ± SD. To compare each mean with every other mean, a one-way ANOVA with Tukey’s multiple comparison was used to analyze statistical significance between means (p < 0.05 [*], p < 0.01 [**], p < 0.001 [***]).

Nakkina S.P., Gitto S.B., Pandey V., Parikh J.G., Geerts D., Maurer H.C., Olive K.P., Phanstiel O I.V, & Altomare D.A. (2021). Differential Expression of Polyamine Pathways in Human Pancreatic Tumor Progression and Effects of Polyamine Blockade on Tumor Microenvironment. Cancers, 13(24), 6391.

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CCT2 Expression in Carcinoma Tissue Microarrays

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Tissue microarrays (TMAs) used in this study were obtained from US Biomax, Inc. Their catalog numbers are as follows: CO484a (colonic carcinoma), PR803b and PR631 (prostate carcinoma), BC03118 (hepatocellular carcinoma), LC726b, LC802a, LC802c, and BC041115a (lung carcinomas) and BN501 (normal tissue array). Each TMA contained varied numbers of patient tissue cores as well as normal tissue corresponding to the specific cancer type being analyzed. Please refer to Supplementary Tables 1, 2 and 5 for the number of samples per cancer type. Information about the tissue type, TNM, score, tumor grade, and stage were provided with the samples. TMAs were stained for CCT2 using anti-CCTβ antibody (LS-B4861; LifeSpan Biosciences). TMA LC802c was stained in parallel for CCT2 and Stat3 (anti-Stat3 antibody ab32500; Abcam). Primary antibodies were diluted 1:100 in Antibody Diluent (Leica). Staining of tissue arrays was performed by a Bond-Max Immunostainer (Leica), with an epitope retrieval buffer of EDTA pH 9.0 (Leica). Polymer Refine Detection reagents (Leica) were used, which include a hematoxylin counterstain. Image acquisition and scoring of staining was performed by a surgical pathologist as previously published [31 (link)].

Carr A.C., Khaled A.S., Bassiouni R., Flores O., Nierenberg D., Bhatti H., Vishnubhotla P., Manuel J.P., Santra S, & Khaled A.R. (2017). Targeting chaperonin containing TCP1 (CCT) as a molecular therapeutic for small cell lung cancer. Oncotarget, 8(66), 110273-110288.

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Immunohistochemical Analysis of CCTβ in Metastatic Breast Cancer

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Lung and liver metastases were induced as described in Supplemental Methods. Mouse organs were harvested and fixed as previously described (23 (link)). Tissues were analyzed using anti-CCTβ primary antibody (LifeSpan Biosciences). Staining of tissue arrays was performed by a Bond-Max Immunostainer (Leica), with an epitope retrieval buffer of EDTA pH 9.0 and Polymer Refine Detection reagents (Leica). Sequential tissue sections were stained with hematoxylin and eosin reagents (Leica). Human breast cancer tissue arrays were purchased from US Biomax (BR1002a, BR10010b, BR963a, and HBre-Duc150-Sur01). Information about tissue type, tumor grade, and receptor status were provided. Array HBre-Duc150-Sur01 also provided information on survival/deceased status of the patient, as well as duration of monitoring in months. Tissues were stained for CCTβ as described above. Scoring of CCTβ staining was done by a pathologist (Dr. Amr Khaled) following the guidelines in Supplemental Fig. 1.

Bassiouni R., Nemec K., Iketani A., Flores O., Showalter A., Khaled A.S., Vishnubhotla P., Sprung RW J.r., Kaittanis C., Perez J.M, & Khaled A.R. (2016). Chaperonin Containing-TCP-1 Protein Level in Breast Cancer Cells Predicts Therapeutic Application of a Cytotoxic Peptide. Clinical cancer research : an official journal of the American Association for Cancer Research, 22(17), 4366-4379.

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