Biosystems refine detection kit

Manufactured by Leica

The Leica Biosystems Refine Detection Kit is a reagent system designed for use in immunohistochemistry (IHC) and in situ hybridization (ISH) procedures. The kit provides the necessary components for the visualization of target analytes in tissue samples.

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Lab products found in correlation

Bond 3, by Leica (3 mentions) Clone d2p2f, by Leica (2 mentions) Antibody for cd14, by Cell Signaling Technology (2 mentions) Aperio versa digital pathology scanner, by Leica (2 mentions) Primary antibody diluent, by Leica (1 mentions) Ix81 inverted microscope, by Olympus (1 mentions) Bond automated staining platform, by Leica (1 mentions) Arg 1, by Abcam (1 mentions) Phospho erk, by Cell Signaling Technology (1 mentions) Bond polymer refine detection kit, by Leica (1 mentions) Cd11b, by Abcam (1 mentions) Bond rx, by Leica (1 mentions) Af1865, by R&D Systems (1 mentions) Alexafluor 488 tyramide, by Thermo Fisher Scientific (1 mentions) Ab6556, by Abcam (1 mentions) Ct adus100, by Chemietek (1 mentions)

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Refine Detection Kit (39 citations) Leica Refine Kit (1 citations)

10 protocols using biosystems refine detection kit

Dual Chromogenic IHC for Microbial Detection

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Single immunohistochemistry was performed on the Leica Bond III automated staining platform using the Leica Biosystems Refine Detection Kit (DS9800). All antibodies were diluted in Leica Primary Antibody Diluent (AR93520). The primary antibodies listed in Table 1 were used at the dilutions indicated. No antigen retrieval was used for any of the primary antibodies.
Dual chromogenic IHC Immunohistochemistry was performed sequentially on the Leica Bond III automated staining platform using the Leica Biosystems Refine Detection Kit (DS9800) and Leica Biosystems Refine Detection Kit (DS9390). All antibodies were diluted in Leica Primary Antibody Diluent (AR93520). The primary antibodies listed in Table 1 were used at the dilutions indicated. For one pair of antibodies, Candida albicans was visualized via 3, 3'-diaminobenzidine (DAB) and S. mutans was visualized via alkaline phosphatase (AP). For another antibody pair, fungal β-D glucan was visualized via DAB and gram-positive bacteria LTA was visualized via AP. For a third antibody pair, gram-negative endotoxin was visualized via DAB and gram-positive bacteria LTA was visualized via AP. For a fourth antibody pair, β-D glucan was visualized via DAB and gram-negative endotoxin was visualized via AP. The coverslips were then mounted and the slides were visualized using an Olympus IX81 inverted microscope.

Sulyanto R.M., Beall C.J., Ha K., Montesano J., Juang J., Dickson J.R., Hashmi S.B., Bradbury S., Leys E.J., Edgerton M., Ho S.P, & Griffen A.L. (2024). Fungi and bacteria occupy distinct spatial niches within carious dentin. PLoS pathogens, 20(5).

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Immunohistochemistry Staining Protocol

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All IHC was performed on the Leica Bond III automated staining platform. Anti-CD3 (A0452, clone F7.2.38, Dako, Santa Clara, CA) was run at 1:250 dilution using the Leica Biosystems Refine Detection Kit with EDTA antigen retrieval. Anti-CD20 (M0755, clone L26, Dako) was run at 1:500 dilution using the Leica Biosystems Refine Detection Kit with citrate antigen retrieval. Anti-VEGF (ab52917, clone EP1176Y, Abcam) was run at 1:100 dilution using the Leica Biosystems Refine Detection Kit with EDTA antigen retrieval. Anti-F4/80 (MCA497GA, clone CI:A3-1, Serotec, Hercules, CA) was run at 1:5000 dilution using the Leica Biosystems Refine Detection Kit with enzymatic antigen retrieval.

Lesch B.J., Tothova Z., Morgan E.A., Liao Z., Bronson R.T., Ebert B.L, & Page D.C. (2019). Intergenerational epigenetic inheritance of cancer susceptibility in mammals. eLife, 8, e39380.

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Quantifying Tumor-Associated Macrophages in Ovarian Cancer

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TAM density was measured using the NHS/NHSII ovarian tumor tissue microarrays as described previously (18 (link)). In brief, for each ovarian cancer paraffin-embedded tissue block containing representative tumor sample, three cores with 0.6mm diameter were embedded in a tissue microarray. CD68 (marker for total TAMs) and CD163 (marker for M2-type TAMs) were stained using immunohistochemistry (IHC) performed on the Leica Bond automated staining platform. Antibody CD68 from Dako catalogue # M0876 clone PG-M1 was run at 1:500 dilution using the Leica Biosystems Refine Detection Kit with CITRATE antigen retrieval. Antibody CD163 from Vector catalogue # VP-C374 clone 10D6 was run at 1:250 dilution using the Leica Biosystems Refine Detection Kit with EDTA antigen retrieval. CD68 and CD163 stains were evaluated in a semiquantitative manner by a gynecologic pathologist. CD68 and CD163 densities were scored separately for tumor stroma and epithelium as: none (1), low (2; <10% of cells, scattered), moderate (3; <10% of cells, with aggregation—at least three aggregates of three macrophages), and high (4; >10% of cells macrophages or an area of confluent macrophages). Tumors were classified as high density (i.e., CD68 high or CD163 high) when the sum of the epithelium and stromal scores were greater than 4 or low density (i.e. CD68 low or CD163 low) when the sum of scores was 4 or less.

Sasamoto N., Wang T., Townsend M.K., Hecht J.L., Eliassen A.H., Song M., Terry K.L., Tworoger S.S, & Harris H.R. (2021). Prospective analyses of lifestyle factors related to energy balance and ovarian cancer risk by infiltration of tumor associated macrophages. Cancer epidemiology, biomarkers & prevention : a publication of the American Association for Cancer Research, cosponsored by the American Society of Preventive Oncology, 30(5), 920-926.

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Quantifying STING and phospho-TBK1 in Brain Tumors

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Brain tumor TMAs (GL2082) were purchased from US Biomax, Inc., and IHC staining for STING and phospho-TBK1 was performed on the Leica Bond III automated staining platform as previously published (53 ). The antibody for phospho-TBK1 (Cell Signaling Technology #5483, clone D52C2) was run at 1:50 dilution with the Leica Biosystems Refine Detection Kit with ethylenediaminetetraacetic acid (EDTA) antigen retrieval. The antibody for STING (Cell Signaling Technology #13647, clone D2P2F) was run at 1:50 dilution with the Leica Biosystems Refine Detection Kit with citrate antigen retrieval. IHC staining was quantified in QuPath software (0.2.0-m4). The positive pixel detection analysis was used with default settings for 3,3′-diaminobenzidine staining to detect and quantify positive pixels in each of three individual, randomly selected fields per tumor, which were then averaged.

Berger G., Knelson E.H., Jimenez-Macias J.L., Nowicki M.O., Han S., Panagioti E., Lizotte P.H., Adu-Berchie K., Stafford A., Dimitrakakis N., Zhou L., Chiocca E.A., Mooney D.J., Barbie D.A, & Lawler S.E. (2022). STING activation promotes robust immune response and NK cell–mediated tumor regression in glioblastoma models. Proceedings of the National Academy of Sciences of the United States of America, 119(28), e2111003119.

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Histological and Immunohistochemical Analysis of Thyroid Tissues

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Thyroid tissues were fixed in 4% paraformaldehyde, embedded in paraffin, sectioned, and stained with hematoxylin and eosin (H&E). Histological diagnosis was performed by thyroid pathologists (R.G. and B.X.) blinded to the genotype and the treatment status of each animal. Immunohistochemistry (IHC) was performed on the Leica Bond III automated staining platform using the Leica Biosystems Refine Detection Kit. The following antibodies were used for IHC: phospho-Erk (Cell Signaling Technology (CST), cat. #4370), Ki-67 (CST, cat. #12202), CD11b (Abcam, cat. #ab133357), F4/80 (CST, cat. #70076), Arg1 (Abcam, cat. #ab 93668) and Pax8 (Proteintech, cat. #10336). The secondary antibodies were part of the Leica Bond Polymer Refine Detection Kit (Catalog No: DS9800). QuPath (Version 0.3.0, (26 (link))) was used for H/E slide visualization and the quantification of DAB (3,3JUNK-Diaminobenzidine) staining on IHC slides. Tumor boundaries were delineated, and the positive cell detection command was applied to the tumor area, applying default settings and a single intensity threshold of 0.2 to quantify the percentage of positive DAB-stained cells.

Landa I., Thornton C.E., Xu B., Haase J., Krishnamoorthy G.P., Hao J., Knauf J.A., Herbert Z.T., Blasco M.A., Ghossein R, & Fagin J.A. (2023). Telomerase reactivation induces progression of mouse BrafV600E-driven thyroid cancers without telomere lengthening. bioRxiv.

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Multiplexed Immunofluorescence Analysis of SARS-CoV-2

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Formalin fixed and paraffin embedded lung parenchymal samples were stained for SARS-CoV-2 nucleocapsid (N), ASC, and CD14 and IF was analyzed on the Leica Bond RX automated staining platform using the Leica Biosystems Refine Detection Kit (Leica). The antibody for SARS Nucleocapsid (Novus) was run with citrate antigen retrieval and tagged with Alexa Fluor 488 Tyramide (Life). Following citrate stripping, the antibody for CD14 (Cell Signaling) was incubated and tagged with Alexa Fluor 594 Tyramide (Life). Following EDTA stripping, staining for ASC (Santa Cruz) was analyzed using antibody tagged with Alexa Fluor 647 Tyramide (Life). Samples were counterstained with DAPI. Slides were scanned using an Aperio Versa Digital Pathology Scanner (Leica) and analyzed with Aperio ImageScope v12.4.3 software (Leica). Slides were also analyzed by confocal microscopy as described above.

Junqueira C., Crespo Â., Ranjbar S., Lewandrowski M., Ingber J., de Lacerda L.B., Parry B., Ravid S., Clark S., Ho F., Vora S.M., Leger V., Beakes C., Margolin J., Russell N., Kays K., Gehrke L., Adhikari U.D., Henderson L., Janssen E., Kwon D., Sander C., Abraham J., Filbin M., Goldberg M.B., Wu H., Mehta G., Bell S., Goldfeld A.E, & Lieberman J. (2021). SARS-CoV-2 infects blood monocytes to activate NLRP3 and AIM2 inflammasomes, pyroptosis and cytokine release. Research Square.

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Multiplex Immunofluorescence Imaging of SARS-CoV-2 in Lung Tissue

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Formalin-fixed and paraffin-embedded lung parenchymal samples were stained for SARS-CoV-2 N, ASC and CD14, and immunofluorescence was analysed on the Leica Bond RX automated staining platform using the Leica Biosystems Refine Detection Kit (Leica). The antibody for SARS nucleocapsid (Novus) was run with citrate antigen retrieval and tagged with Alexa Fluor 488 Tyramide (Life). After citrate stripping, the antibody for CD14 (Cell Signaling) was incubated and tagged with Alexa Fluor 594 Tyramide (Life). After EDTA stripping, staining for ASC (Santa Cruz) was analysed using antibodies tagged with Alexa Fluor 647 Tyramide (Life). EDTA stripping was performed before anti-CD31 or anti-E-cadherin staining tagged to Alexa Fluor 555 Tyramide (Life). The samples were counterstained with DAPI. The slides were scanned using the Aperio Versa Digital Pathology Scanner (Leica) and analysed using Aperio ImageScope v.12.4.3 (Leica). The slides were also analysed by confocal microscopy as described above.

Junqueira C., Crespo Â., Ranjbar S., de Lacerda L.B., Lewandrowski M., Ingber J., Parry B., Ravid S., Clark S., Schrimpf M.R., Ho F., Beakes C., Margolin J., Russell N., Kays K., Boucau J., Adhikari U.D., Vora S.M., Leger V., Gehrke L., Henderson L.A., Janssen E., Kwon D., Sander C., Abraham J., Goldberg M.B., Wu H., Mehta G., Bell S., Goldfeld A.E., Filbin M.R, & Lieberman J. (2022). FcγR-mediated SARS-CoV-2 infection of monocytes activates inflammation. Nature, 606(7914), 576-584.

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Immunohistochemical Analysis of Phospho-TBK1 and STING in Brain Tumor Tissue

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Brain tumor and brain metastasis tissue microarrays (GL2082, GL861) were purchased from US Biomax, Inc and IHC was performed on the Leica Bond III automated staining platform. The antibody for phospho-TBK1 (Cell Signaling Technology #5483, clone D52C2) was run at 1:50 dilution using the Leica Biosystems Refine Detection Kit with EDTA antigen retrieval. The antibody for STING (Cell Signaling Technology #13647, clone D2P2F) was run at 1:50 dilution using the Leica Biosystems Refine Detection Kit with citrate antigen retrieval. Staining was visually scored in a binary manner (presence/absence) in endothelial cells identified using the hematoxylin counterstain marking a circumferential layer of nuclei surrounding red blood cell fragments. These results were confirmed by a board-certified anatomic pathologist (NRM), who also quantified infiltrating lymphocytes by morphology on hematoxylin-counterstained per high power field (HPF = 40× objective), averaged across confidently identified endothelial lumens in 1–4 HPF per specimen. Average tumor infiltrating lymphocytes per HPF was compared for pTBK1+ and pTBK1− blood vessels in each tumor specimen.

Campisi M., Sundararaman S.K., Shelton S.E., Knelson E.H., Mahadevan N.R., Yoshida R., Tani T., Ivanova E., Cañadas I., Osaki T., Lee S.W., Thai T., Han S., Piel B.P., Gilhooley S., Paweletz C.P., Chiono V., Kamm R.D., Kitajima S, & Barbie D.A. (2020). Tumor-Derived cGAMP Regulates Activation of the Vasculature. Frontiers in Immunology, 11, 2090.

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Intramuscular AAVrh32.33 Gene Delivery

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Adult C57BL/6J or Myd88^−/− mice were injected intramuscularly (i.m.) with 50 μl PBS or AAVrh32.33 single-stranded viruses (1 × 10¹⁰ or 1 × 10¹¹ vg per animal) encoding GFP or hFIX in the left quadriceps muscle. 21 d later, the animals were sacrificed and a portion of the quadriceps was fixed in 10% formalin overnight and transferred to 70% ethanol. For histology, muscle samples were processed at Dana-Farber/Harvard Cancer Center Specialized Histopathology Services and Beth Israel Deaconess Medical Center histology core facilities, embedded in paraffin, and stained for CD8 (1:500, D4W2Z, CST) and granzyme B (1:500, polyclonal, catalog # AF1865, R&D). Antibodies were tagged with AlexaFluor 488 Tyramide or AlexaFluor 647 Tyramide (B40957 and B40958, Thermo Fisher). All immunohistochemistry was performed on the Leica Bond automated staining platform using the Leica Biosystems Refine Detection Kit with citrate antigen retrieval. Muscle sections also were stained for GFP (1:800, polyclonal, catalog # ab6556, Abcam) using the Leica Biosystems Refine Detection Kit (DAB chromogen) with EDTA antigen retrieval. For rh32.33.FIX injected mice, plasma samples were obtained 14 d, 28 d, 42 d and 60 d after 1 × 10¹¹ vg AAV administration and hFIX expression was measured by ELISA as previously described.

Chan Y.K., Wang S.K., Chu C.J., Copland D.A., Letizia A.J., Verdera H.C., Chiang J.J., Sethi M., Wang M.K., Neidermyer WJ J.r., Chan Y., Lim E.T., Graveline A.R., Sanchez M., Boyd R.F., Vihtelic T.S., Inciong R.G., Slain J.M., Alphonse P.J., Xue Y., Robinson-McCarthy L.R., Tam J.M., Jabbar M.H., Sahu B., Adeniran J.F., Muhuri M., Tai P.W., Xie J., Krause T.B., Vernet A., Pezone M., Xiao R., Liu T., Wang W., Kaplan H.J., Gao G., Dick A.D., Mingozzi F., McCall M.A., Cepko C.L, & Church G.M. (2021). Engineering adeno-associated viral vectors to evade innate immune and inflammatory responses. Science translational medicine, 13(580), eabd3438.

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Quantitative STING and phospho-IRF3 IHC

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IHC staining for STING and phospho-IRF3 was performed on the Leica Bond III automated staining platform. The antibody specific for STING (Cell Signaling Technology #13647, clone D2P2F) was run at 1:50 dilution using the Leica Biosystems Refine Detection Kit (#DS9800) with citrate antigen retrieval. The antibody specific for phospho-IRF3 (Cell Signaling Technology #29047, clone D6O1M) was run at 1:100 dilution using the Leica Biosystems Refine Detection Kit (# DS9800) with EDTA antigen retrieval (n=31). This was optimized from a range of dilutions from 1:50 to 1:200 and comparison of citrate vs. EDTA antigen retrieval on MPM cell lines treated in vitro with 50 μM ADU-S100 (Chemietek #CT-ADUS100) for 24-hours prior to paraformaldehyde fixation and paraffin embedding. STING IHC staining was quantified using the QuPath software (version 0.2.3) (21 (link)). Positive Pixel Detection analysis was used with default settings for DAB staining to detect and quantify positive pixels in each of three individual, randomly selected fields per tumor, which were then averaged. Phospho-IRF3 levels were too low to quantify systematically with this software.

Knelson E.H., Ivanova E.V., Tarannum M., Campisi M., Lizotte P.H., Booker M.A., Ozgenc I., Noureddine M., Meisenheimer B., Chen M., Piel B., Spicer N., Obua B., Messier C.M., Shannon E., Mahadevan N.R., Tani T., Schol P.J., Lee-Hassett A.M., Zlota A., Vo H.V., Ha M., Bertram A.A., Han S., Thai T.C., Gustafson C.E., Venugopal K., Haggerty T.J., Albertson T.P., Hartley A.V., Eser P.O., Li Z.H., Cañadas I., Vivero M., De Rienzo A., Richards W.G., Abu-Yousif A.O., Appleman V.A., Gregory R.C., Parent A., Lineberry N., Smith E.L., Jänne P.A., Miret J.J., Tolstorukov M.Y., Romee R., Paweletz C.P., Bueno R, & Barbie D.A. (2022). Activation of tumor-cell STING primes NK-cell therapy. Cancer immunology research, 10(8), 947-961.

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