Hrp conjugated secondary antibody

Manufactured by Roche

The HRP-conjugated secondary antibody is a laboratory reagent used in immunoassay techniques, such as Western blotting and ELISA, to detect and quantify target proteins. The HRP (horseradish peroxidase) enzyme conjugated to the secondary antibody enables the detection and visualization of the target analyte through a colorimetric or chemiluminescent reaction.

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Omni Map anti-rabbit HRP-conjugated secondary antibody (Multimer HRP) (3 citations)

5 protocols using hrp conjugated secondary antibody

Immunohistochemical Analysis of Leiomyoma Tissues

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IHC was performed to evaluate the expression of TGFBR2 and IGF2BP1 in 16 leiomyoma tissues. Tissues were fixed in formalin and embedded in paraffin, and 3-μm-thick paraffin sections were obtained. IHC was performed using an automated immunohistochemical stainer (Ventana Medical Systems, Inc., Tucson, AZ, USA) according to the manufacturer’s protocol. The sections were deparaffinized and pretreated with a cell-conditioning solution (CC1, Ventana), followed by UV irradiation to abrogate the endogenous hydroperoxidase activity. The primary antibodies were diluted in Dako antibody diluent (Dako Cytomation, Glostrup, Denmark) with background-reducing components and used under the following dilutions: TGFBR2 (1:100, ab61213, Abcam, Franklin Lakes, NJ, USA) and IGF2BP1 (1:100, ab82968, Abcam,). The sections were incubated with primary antibodies at room temperature for 32 min, and hybridized with HRP-conjugated secondary antibody (Ventana) for 8 min. The reaction was developed with diaminobenzidine (DAB; Dako) for 5 min and the slides were counterstained with hematoxylin II (Ventana) for 4 min and bluing reagent (Ventana) for 4 min. The sections were observed under light microscope (BX50, Olympus, Japan).

Kim M., Kang D., Kwon M.Y., Lee H.J, & Kim M.J. (2022). MicroRNAs as potential indicators of the development and progression of uterine leiomyoma. PLoS ONE, 17(5), e0268793.

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In Situ Hybridization of microRNA in FFPE Tissue

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Deparrafinization and rehydration of the formalin-fixed, paraffin-embedded tissue sections used xylene and an ethanol dilution series. Sections of tissue sections were digested with 15 µg/mL proteinase K for 20 minutes at room temperature and loaded onto Ventana Discovery Ultra for in situ hybridization analysis. Tissue slides were incubated with double-DIG labeled mercury LNA microRNA probe (exiqon) for two hours at 55°C. Three percent H₂O₂ was used to inactivate endogenous peroxidases. Following polyclonal anti-DIG antibody and HRP-conjugated secondary antibody (Ventana) incubation, tyramine-conjugated fluorochrome (TSA) reaction was performed for twelve minutes. Sequential TSA rounds were performed for the detection of proteins using the same protocol. Slides were mounted with antifading ProLong Gold Solution (Life Technologies).

Rupaimoole R., Ivan C., Yang D., Gharpure K.M., Wu S.Y., Pecot C.V., Previs R.A., Nagaraja A.S., Armaiz-Pena G.N., McGuire M., Pradeep S., Mangala L.S., Rodriguez-Aguayo C., Huang L., Bar-Eli M., Zhang W., Lopez-Berestein G., Calin G.A, & Sood A.K. (2016). Hypoxia-upregulated microRNA-630 targets Dicer, leading to increased tumor progression. Oncogene, 35(33), 4312-4320.

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Immunohistochemical Analysis of Cytokine Expression

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Tissues were fixed in formalin, embedded in paraffin, and cut into 3-µm-thick sections. Immunohistochemical staining (IHC) was performed using an automated immunohistochemical stainer (Ventana, Tucson, AZ, USA) according to the manufacturer’s protocol. The sections were deparaffinized, pretreated with Cell Conditioning 1 solution (Ventana), and subjected to ultraviolet irradiation to abolish endogenous hydroperoxidase activity. The primary antibodies were diluted in Dako antibody diluent (Dako Cytomation, Glostrup, Denmark) with background-reducing components and were used at the following dilutions: IL-12 (rabbit, 1:100; Abcam, Franklin Lakes, NJ, USA), IL-2 (rabbit, 1:250; Abcam), IFN-γ (rabbit, 1:1,000; Abcam), IL-23 (rabbit, 1:200; Abcam), IL-17A (mouse, 1:100; Abcam), and IL-22 (rabbit, 1:300; Abcam). The sections were incubated with primary antibodies at room temperature for 32 minutes and then hybridized with an HRP-conjugated secondary antibody (Ventana) for 8 minutes. The reaction was developed by incubating with diaminobenzidine (DAB; Dako Cytomation) for 5 minutes, and the slides were counterstained with hematoxylin II (Ventana) for 4 minutes and with bluing reagent (Ventana) for 4 minutes. The sections were observed under a light microscope (BX50; Olympus, Tokyo, Japan).

Ju H.J., Park H.J., Choi I.H., Lee K.H., Kwon M.Y, & Park C.J. (2022). Comparison of Th1 and Th17 Inflammatory Cytokine Profiles Between Chronic Plaque and Acute Guttate Psoriasis. Annals of Dermatology, 34(3), 200-205.

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In Situ Hybridization for miRNA and lncRNA Detection

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The formalin-fixed paraffin embedded tissue sections were dewaxed in xylenes and rehydrated through an ethanol dilution series⁶⁰. Tissue sections were digested with 5 μg ml⁻¹ proteinase K for 15 min at RT, and were then loaded onto Ventana Discovery Ultra for in situ hybridization analysis. The tissue slides were incubated with double-DIG labeled mercury LNA probe (Exiqon) for 2 h. The slides were treated with 3% H₂O₂ to inactivate endogenous peroxidase. Followed by a rabbit polyclonal anti-DIG antibody (Sigma, Cat.#: D7782, 1:10,000 dilution) and HRP conjugated secondary antibody (Ventana, Cat.#: 760–4311, ready to use) incubation, tyramine-conjugated fluorochrome (TSA) reaction was performed for 12 min. Finally, the slides were mounted with antifading ProLong Gold Solution (Life Technologies). For the chromogenic detection of miRNAs, miRNA signal was recognized by anti-DIG antibody, and alkaline phosphatase (AP) conjugated second antibody (Ventana, Cat.#: 760–4314, ready to use) using NBT-BCIP as the substrate. The following probes were used: CCNE2: 5′-TGCTCTTCGGTGGTGTCATAA-3’ and uc.339: 5′-AGATGGAGGATCGGTGTGAAA-3′. For U6 control probe # 99002-15 (Exiqon) was used. For miR-339-3p, probe # 38578-15 (Exiqon) and for miR-663b-3p, probe # 21359-15 (Exiqon) were used. The uc339 signal in ISH was amplified by anti-rab-HRP antibody and Tyramide signal amplification (TSA) system.

Vannini I., Wise P.M., Challagundla K.B., Plousiou M., Raffini M., Bandini E., Fanini F., Paliaga G., Crawford M., Ferracin M., Ivan C., Fabris L., Davuluri R.V., Guo Z., Cortez M.A., Zhang X., Chen L., Zhang S., Fernandez-Cymering C., Han L., Carloni S., Salvi S., Ling H., Murtadha M., Neviani P., Gitlitz B.J., Laird-Offringa I.A., Nana-Sinkam P., Negrini M., Liang H., Amadori D., Cimmino A., Calin G.A, & Fabbri M. (2017). Transcribed ultraconserved region 339 promotes carcinogenesis by modulating tumor suppressor microRNAs. Nature Communications, 8, 1801.

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In Situ Hybridization of microRNA in FFPE Tissue

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