Ezprep buffer

Manufactured by Roche

Sourced in United States

EZPrep buffer is a laboratory product designed for nucleic acid extraction and purification. It is a buffer solution used in the preparatory steps of molecular biology techniques, such as DNA or RNA isolation. The core function of the EZPrep buffer is to facilitate the release, stabilization, and preparation of nucleic acids from various sample types prior to further analysis or processing.

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Lab products found in correlation

Cc1 buffer, by Roche (24 mentions) Discovery xt processor, by Roche (20 mentions) Dab detection kit, by Roche (14 mentions) Permount, by Thermo Fisher Scientific (12 mentions) Biotinylated goat anti rabbit igg, by Vector Laboratories (12 mentions) Avidin biotin blocking, by Roche (10 mentions) Biotinylated horse anti mouse igg, by Vector Laboratories (5 mentions) Pannoramic viewer, by 3DHISTECH (4 mentions) Hematoxylin, by Roche (4 mentions) 4 6 diamidino 2 phenylindole (dapi), by Merck Group (4 mentions) Tyramide alexa fluor 488, by Thermo Fisher Scientific (3 mentions) Streptavidin hrp d, by Roche (3 mentions) Dab map kit, by Roche (3 mentions) Alkaline blue detection kit, by Roche (3 mentions) Synaptophysin, by Vector Laboratories (2 mentions) Itgb4, by Cell Signaling Technology (2 mentions) Biotinylated horse anti rabbit antibody, by Vector Laboratories (2 mentions) Itgb4, by Vector Laboratories (2 mentions) Vimentin, by Vector Laboratories (2 mentions) Paraformaldehyde (pfa), by Thermo Fisher Scientific (2 mentions)

35 protocols using ezprep buffer

Tissue Processing and Immunohistochemistry for Xenograft and Transgenic Mouse Samples

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For xenograft samples and pancreatic tissue from KPC mice, dissected tissues were fixed immediately after removal in 10% buffered formalin solution for a maximum of 24 h at room temperature before being dehydrated and paraffin-embedded under a vacuum. The tissue sections were deparaffinized with EZ Prep buffer (Ventana Medical Systems, Santa Clara, USA). Antigen retrieval was performed with CC1 buffer (Ventana Medical Systems), and sections were blocked for 30 min with Background Buster solution (Innovex, Lincoln, RI, USA). IHC detection was performed using the Discovery XT processor (Ventana Medical Systems). All tumor tissues were harvested from mice and fixed in 4% PFA overnight. Fixed tissues were dehydrated, embedded in paraffin, and sliced into 3-µm sections. The tissue sections were deparaffinized with EZ Prep buffer, and antigen retrieval was performed with CC1 buffer and heat treatment in citrate buffer at pH 6.0 (Ribo CC, Ventana Medical Systems). Tumor sections were incubated with the indicated primary antibodies and detection was performed with a DAB detection kit (Ventana Medical Systems) according to the manufacturer’s instructions, followed by counterstaining with hematoxylin (Ventana Medical Systems). Images were obtained using Vectra Polaris (PerkinElmer).

Lee D.E., Yoo J.E., Kim J., Kim S., Kim S., Lee H, & Cheong H. (2020). NEDD4L downregulates autophagy and cell growth by modulating ULK1 and a glutamine transporter. Cell Death & Disease, 11(1), 38.

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Immunohistochemical Staining of Paraffin Sections

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Immunohistochemical and immunofluorescent staining of paraffin-embedded sections was performed by using a Discovery XT processor (Ventana Medical Systems). Tissue sections were deparaffinized with EZPrep buffer (Ventana Medical Systems) and subjected to antigen retrieval with CC1 buffer (Ventana Medical Systems). Sections were blocked for 30 minutes with Background Buster solution (Innovex) and for 8 minutes with avidin-biotin blocking (Ventana Medical Systems). Sections were incubated with anti-AR (Abcam, ab133273 1 μg/mL); ITGB4 (Cell Signaling, cat# 14803, 0.5 μg/mL); Vimentin (Cell Signaling, cat# 5741, 0.5 μg/mL); and synaptophysin (Abcam, ab32127, 1 μg/mL) antibodies for 5 hours followed by biotinylated horse anti-rabbit antibodies (Vector Labs, cat# PK6101) at 1:200 dilution (for AR, ITGB4, Vimentin) or HRP-conjugated goat anti-rabbit (PI-1000) antibodies at 1:250 dilution (for synaptophysin) for 60 minutes. Detection was performed with DAB detection kit (Ventana Medical Systems) according to manufacturer instruction. Slides were counterstained with hematoxylin and mounted using Permount (Fisher Scientific).

Han H., Wang Y., Curto J., Gurrapu S., Laudato S., Rumandla A., Chakraborty G., Wang X., Chen H., Jiang Y., Kumar D., Caggiano E.G., Capogiri M., Zhang B., Ji Y., Maity S.N., Hu M., Bai S., Aparicio A.M., Efstathiou E., Logothetis C.J., Navin N., Navone N.M., Chen Y, & Giancotti F.G. (2022). Mesenchymal and stem-like prostate cancer linked to therapy-induced lineage plasticity and metastasis. Cell reports, 39(1), 110595.

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Immunohistochemical Analysis of IL33 Expression

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Tissues were fixed in paraformaldehyde (Fisher Scientific) for 24 hours and embedded in paraffin. The tissue sections were deparaffinized with EZPrep buffer (Ventana Medical Systems), then antigen retrieval was performed with CC1 buffer (Ventana Medical Systems). Sections were blocked for 30 minutes with Background Buster solution (Innovex), followed by avidin-biotin blocking for 8 minutes (Ventana Medical Systems). Mouse IL33 (AF3626, R&D Systems), mouse smooth muscle actin (Abcam), and human IL33 (AF3625, R&D Systems) antibodies were applied, and sections were incubated for 4 hours, followed by a 60-minute incubation with biotinylated rabbit anti-goat IgG (Vector labs), or biotinylated goat anti-rabbit IgG (Vector labs) at 1:200 dilution. Detection was performed with DAB detection kit (Ventana Medical Systems) according to the manufacturer’s instructions. Any section containing cells demonstrating cytoplasmic or nuclear positivity for IL33 was designated to have positive staining. Slides were counterstained with Masson’s trichrome, or hematoxylin, and eosin,and cover-slipped with Permount (Fisher Scientific).All histologic sections were evaluated by an independent PDAC pathologist.

Moral J.A., Leung J., Rojas L.A., Ruan J., Zhao J., Sethna Z., Ramnarain A., Gasmi B., Gururajan M., Redmond D., Askan G., Bhanot U., Elyada E., Park Y., Tuveson D.A., Gönen M., Leach S.D., Wolchok J.D., DeMatteo R.P., Merghoub T, & Balachandran V.P. (2020). ILC2s amplify PD-1 blockade by activating tissue-specific cancer immunity. Nature, 579(7797), 130-135.

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Immunohistochemical Detection of Cleaved Caspase-3

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SU-DH-L-6 cells were stained using a Discovery XT processor (Ventana Medical Systems) in the Memorial Sloan Kettering Cancer Center Molecular Cytology Core Facility. The slides were deparaffinized with EZPrep buffer (Ventana Medical Systems), antigen retrieval was performed with CC1 buffer (Ventana Medical Systems), and sections were blocked for 30 min with Background Buster solution (Innovex) followed by Avidin/biotin blocking for 8 min. Anti-Cleaved Caspase 3 (9661, 0.1 μg/mL; Cell Signaling) antibodies were applied and sections were incubated for 5 h, followed by 60 min incubation with biotinylated goat anti-rabbit IgG (PK6101; Vector Laboratories) at 1:200 dilution. The detection was performed with a DAB detection kit (Ventana Medical Systems) according to the manufacturer’s instructions. Slides were counterstained with hematoxylin and coverslipped with Permount (Fisher Scientific). Tissue sections were digitally scanned using Pannoramic Flash (3DHistech) with a 20×/0.8 N.A. objective. Slides were then reviewed using the Pannoramic Viewer (3DHistech) software and then images were taken and exported into TIFF files at both 20× and 51× (1:1) magnification.

Liu Y., Mondello P., Erazo T., Tannan N.B., Asgari Z., de Stanchina E., Nanjangud G., Seshan V.E., Wang S., Wendel H.G, & Younes A. (2018). NOXA genetic amplification or pharmacologic induction primes lymphoma cells to BCL2 inhibitor-induced cell death. Proceedings of the National Academy of Sciences of the United States of America, 115(47), 12034-12039.

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Multiplexed Immunofluorescence Staining Protocol

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Automated immunofluorescence (IF) staining was performed at the
Molecular Cytology Core Facility of Memorial Sloan Kettering Cancer Center
using a Discovery XT processor (Ventana Medical Systems). The tissue
sections were deparaffinized with EZPrep buffer (Ventana Medical Systems),
antigen retrieval was performed with CC1 buffer (Ventana Medical Systems).
Sections were blocked for 30 min with Background Buster solution (Innovex),
followed by avidin-biotin blocking for 8 min (Ventana Medical Systems).
Multiplexed immunofluorescence stainings were performed as previously
described (Yarilin et al., 2015 (link)).
Staining was performed in the following order: Anti-Claudin-4 (Invitrogen,
catalog #36–4800, 5 μg/ml), anti-Claudin-2 (Invitrogen,
catalog #32–5600, 5 μg/ml), anti-Lysozyme (DAKO, catalog
#A0099, 2 μg/ml). After staining slides were counterstained with DAPI
(Sigma Aldrich, catalog #D9542, 5 μg/ml) for 10 min and coverslipped
with Mowiol mounting reagent. Secondary antibodies used for visualization
were AF488 (Claudin-4), AF594 (Claudin-2), and AF546 (Lysozyme). Slides were
scanned to acquire fluorescence signal.

Marjanovic N.D., Hofree M., Chan J.E., Canner D., Wu K., Trakala M., Hartmann G.G., Smith O., Kim J., Evans K.V., Hudson A., Ashenberg O., Porter C.B., Bejnood A., Subramanian A., Pitter K., Yan Y., Delroy T., Phillips D.R., Shah N., Chaudhary O., Tsankov A., Hollmann T., Rekhtman N., Massion P.P., Poirier J.T., Mazutis L., Li R., Lee J.H., Amon A., Rudin C.M., Jacks T., Regev A, & Tammela T. (2020). Emergence of a high-plasticity cell state during lung cancer evolution. Cancer cell, 38(2), 229-246.e13.

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Cellular Distribution of Fluorescent Colloidal Nanocrystals

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Five immunocompetent C57BL6 mice (5 months old, female) were examined to identify the cellular distribution of fluorophore-labeled fCNC. Each animal received a single intravenous dose of fCNC-AF488 and after 24 h, the heart, kidneys, liver, and spleen were harvested, washed in ice cold PBS, and fixed overnight in 4% paraformaldehyde at 4 °C. Fixed tissue was subsequently embedded in paraffin, and sectioned to obtain 0.005 mm samples. Immunofluorescent (IF) staining was performed as previously described^{17 (link),18 (link)} using a Discovery XT processor (Ventana Medical Systems). Briefly, tissue sections were deparaffinized with EZPrep buffer (Ventana Medical Systems), antigen retrieval was performed with CC1 buffer (Ventana Medical Systems) and sections were blocked for 30 min with Background Buster solution (Innovex). The anti-AF488 antibodies (Molecular Probes, cat. no. A-11094, 5 µg/mL) were applied and sections were incubated for 5 h, followed by 60 min. incubation with biotinylated goat anti-rabbit IgG (Vector labs, cat. no. PK6101, 1:200 dilution)^{15 (link),17 (link),18 (link)}. Slides were counterstained with 4′,6-diamidino-2-phenylindole, (DAPI, Sigma Aldrich, 5 μg/mL) for 10 min. and coverslipped with Mowiol. The tissue distribution fCNC was imaged using anti-AF488 IF staining. Images were acquired using Pannoramic Flash (3D Histech) using a 20 × 0.8NA objective.

Wong S., Alidori S., Mello B.P., Almeida B.A., Ulmert D., Brendel M.B., Scheinberg D.A, & McDevitt M.R. (2021). Fibrillar pharmacology of functionalized nanocellulose. Scientific Reports, 11, 157.

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Immunohistochemical Staining Protocol

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The immunohistochemical detection of indicated antibodies was performed at Molecular Cytology Core Facility of Memorial Sloan Kettering Cancer Center using Discovery XT processor (Ventana Medical Systems). The tissue sections were deparaffinized with EZPrep buffer (Ventana Medical Systems), antigen retrieval was performed with CC1 buffer (Ventana Medical Systems) and sections were blocked for 30 minutes with Background Buster solution (Innovex)(for rabbit, mouse and chicken Abs, for rat and goat Abs it should be 10% normal rabbit serum in PBS+2%BSA). Antibodies were applied and sections were incubated for 5 hours, followed by 60 minutes incubation with biotinylated goat anti-rabbit IgG (Vector labs, cat#PK6101) (biotinylated horse anti-mouse IgG (Vector Labs, cat# MKB-22258); biotinylated goat anti-chicken (Vector Labs, cat#BA-9010); biotinylated rabbit anti-goat IgG (Vector, cat #BA-5000)) at 1:200 dilution. The detection was performed with DAB detection kit (Ventana Medical Systems) according to manufacturer instruction. Slides were counterstained with hematoxylin and coverslipped with Permount (Fisher Scientific). Staining quantification was performed using MetaMorph Software.

Walsh L.A., Roy D.M., Reyngold M., Giri D., Snyder A., Turcan S., Badwe C.R., Lyman J., Bromberg J., King T.A, & Chan T.A. (2014). RECK Controls Breast Cancer Metastasis by Modulating a Convergent, STAT3-dependent Neoangiogenic Switch. Oncogene, 34(17), 2189-2203.

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Mouse Autoantibody Detection by ELISA

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Quantification of serum Ig isotypes was performed by ELISA as previously described^{40 (link)}. Tissue sections from gender matched Rag1^−/− mice were used to detect mouse autoantibodies. Briefly, organs from the Rag1^−/− mice were dissected, fixed with neutral buffered formalin, embedded with paraffin and sectioned. After deparaffinization with EZPrep buffer (Ventana Medical Systems) and antigen retrieval with Cell Conditioning Solution (CC1) (Ventana Medical Systems) the sections were blocked for 30 minutes with Background Buster solution (Innovex), followed by Avidin/Biotin blocking for 8 minutes, mouse serum (1:50 dilution) incubation for 5 hours and biotinylated horse anti-mouse IgG (Vector Labs) incubation for 1 hour. The detection was performed with Streptavidin-HRP (Ventana Medical Systems) followed by incubation with Tyramide Alexa Fluor 488 (Invitrogen). The slides were then counterstained with DAPI (Sigma Aldrich) for 10 minutes, mounted, scanned with a Mirax scanner and visualized with Pannoramic Viewer (3DHISTECH). Scanned images were scored and representative snapshots were processed with Photoshop (Adobe) to switch the green and red channels for presentation purpose.

Feng Y., van der Veeken J., Shugay M., Putintseva E.V., Osmanbeyoglu H.U., Dikiy S., Hoyos B.E., Moltedo B., Hemmers S., Treuting P., Leslie C.S., Chudakov D.M, & Rudensky A.Y. (2015). A mechanism for expansion of regulatory T cell repertoire and its role in self tolerance. Nature, 528(7580), 132-136.

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Immunofluorescent Staining of pErk1/2 and Mac1

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The immunofluorescent staining was performed at Molecular Cytology Core Facility of Memorial Sloan Kettering Cancer Center using Discovery XT processor (Ventana Medical Systems). The tissue sections were deparaffinized with EZPrep buffer (Ventana Medical Systems) and antigen retrieval was performed with CC1 buffer (Ventana Medical Systems). Sections were blocked for 30 min with Background Buster solution (Innovex), followed by avidin-biotin blocking (Ventana Medical Systems) for 8 min. Slides were incubated with anti-pErk1/2 (Cell Signaling, cat# 4370, 1 μg/ml) and anti-Mac1 (abcam, cat#ab133357, 1 μg/ml) for 5 hr, followed by 60 min incubation with biotinylated goat anti-rabbit (Vector Labs, cat# PK6101, 5.75 μg/ml) at 1:200 dilution. The detection was performed with Streptavidin-HRP D (part of DABMap kit, Ventana Medical Systems), followed by incubation with Tyramide Alexa Fluor 488 (Invitrogen, cat# T20922, for pErk1/2) and Tyramide Alexa Fluor 568 (Invitrogen, cat#T20914, for Mac1) prepared according to the manufacturer’s protocol with predetermined dilutions. After staining slides were counterstained with DAPI (Sigma Aldrich, cat# D9542, 5 μg/ml) for 10 min and coverslipped with Mowiol.

Kunimoto H., Meydan C., Nazir A., Whitfield J., Shank K., Rapaport F., Maher R., Pronier E., Meyer S.C., Garrett-Bakelman F.E., Tallman M., Melnick A., Levine R.L, & Shih A.H. (2017). Cooperative Epigenetic Remodeling by TET2 Loss and NRAS Mutation Drives Myeloid Transformation and MEK Inhibitor Sensitivity. Cancer cell, 33(1), 44-59.e8.

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Multiplex Immunofluorescence Staining of Tissue Sections

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The immunofluorescence was performed at the Molecular Cytology Core Facility of Memorial Sloan Kettering Cancer Center using Discovery XT processor (Ventana Medical Systems). The tissue sections were deparaffinized with EZPrep buffer (Ventana Medical Systems), antigen retrieval was performed with CC1 buffer (Ventana Medical Systems). Sections were blocked for 30 min with Background Buster solution (Innovex), followed by avidin-biotin blocking for 8 min (Ventana Medical Systems). Multiplex immunofluorescence stainings were performed as previously described^{53 (link)} (see Supplementary Note). Stained slides were digitized using Pannoramic Flash 250 (3DHistech, Hungary) using 20x/0.8NA objective. Regions of interest were drawn on the scanned images using Pannoramic Viewer (3DHistech, Hungary) and exported into tiff images. ImageJ/FIJI was used to segment DAPI-stained nuclei and count the cells with positive signal. Ki67 was quantified according to the recommendations for breast cancer^{54 (link)}, where the percentage of positively stained nuclei is quantified among the total number of malignant cells as previously described¹³.

Jiménez-Sánchez A., Cybulska P., Mager K.L., Koplev S., Cast O., Couturier D.L., Memon D., Selenica P., Nikolovski I., Mazaheri Y., Bykov Y., Geyer F.C., Macintyre G., Gavarró L.M., Drews R.M., Gill M.B., Papanastasiou A.D., Sosa R.E., Soslow R.A., Walther T., Shen R., Chi D.S., Park K.J., Hollmann T., Reis-Filho J.S., Markowetz F., Beltrao P., Vargas H.A., Zamarin D., Brenton J.D., Snyder A., Weigelt B., Sala E, & Miller M.L. (2020). Unraveling tumor-immune heterogeneity in advanced ovarian cancer uncovers immunogenic effect of chemotherapy. Nature genetics, 52(6), 582-593.

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