Discovery purple kit

Manufactured by Roche

The Discovery Purple Kit is a laboratory equipment product designed for scientific research and analysis purposes. It serves as a core tool for various applications, though its specific intended use is not provided in this factual and unbiased description.

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8 protocols using discovery purple kit

Dual Immunohistochemical Analysis of Stricture Regions

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Serial 4-μm-thick sections from uninflamed and inflamed regions of each block of stricture were taken to perform the following immunohistochemical analyses, as previously described^{44 (link)}. For double stains, tissue specimens were fixed in 10% formalin and embedded in paraffin and 3-μm sections were used for immunohistochemistry.
Immunohistochemistry was performed using VENTANA DISCOVERY ULTRA from Roche. This system allows for automated baking, deparaffinization and cell conditioning. Semiautomatic dual staining was performed sequentially using WT1 at a 1:25 dilution (abcam ab89901) during 60 min. An automated one drop of a prediluted secondary antibody Discovery OMNIMap anti-rabbit-HRP from Roche (760–4310) was used and the signal was obtained using Discovery ChromoMap DAB RUO from Roche (760–2513) (brown signal). PDGFRA (Thermofisher TA804956) was used at a 1:50 dilution during 60 min and after this, secondary antibody (Discovery OMNIMap anti-mouse-NP from Roche (760–4816)) positive signal was obtained using Discovery Purple Kit (760–229)(purple signal). Tissues were counterstained with haematoxylin to visualize the nuclei.

Nayar S., Morrison J.K., Giri M., Gettler K., Chuang L.S., Walker L.A., Ko H.M., Kenigsberg E., Kugathasan S., Merad M., Chu J, & Cho J.H. (2021). A myeloid–stromal niche and gp130 rescue in NOD2-driven Crohn’s disease. Nature, 593(7858), 275-281.

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Immunohistochemical Detection of Y. pestis

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Groups of sterile or infected lice with detectable mCherry signal in the head were transferred to a drop of PBS, their cuticle was punctured 3 times with an insect pin, and lice were transferred to 10% neutral buffered formalin and fixed for at least 24 h. Fixed lice were processed using the VIP-6 Tissue Tek processor (Sakura Finetek USA) and then embedded in paraffin. Blocks were sectioned at 5 μm and every third section was stained with hematoxylin and eosin to identify the desired regions for IHC. Labeling of Y. pestis was performed with a polyclonal α-Y. pestis antibody [54 (link)] (rabbit; 1:4,000) and a secondary α-rabbit-HRP conjugate (Vector Laboratories ImmPRESS-VR Horse anti-rabbit IgG polymer). IHC was performed using the Discovery purple kit and the Roche/Ventana Discovery ULTRA staining platform according to manufacturer’s instructions (Roche Tissue Diagnostics). Slides were counterstained with hematoxylin and Y. pestis appears pink or purple following staining with the Discovery chromogen.

Bland D.M., Long D., Rosenke R, & Hinnebusch B.J. (2024). Yersinia pestis can infect the Pawlowsky glands of human body lice and be transmitted by louse bite. PLOS Biology, 22(5), e3002625.

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Immunohistochemical Analysis of Lung Tumors

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Lungs were fixed in 10% buffered formalin, embedded in paraffin wax and sectioned at 5 mm. For pathological examination sections were stained with hematoxylin and eosin, according to standard procedures. Antibodies used for immunohistochemistry in lung tumor sections included those raised against: γH2AX Ser 139 (Millipore), Ki67 (Master diagnostica), C3A (Cell Signaling Technology), β-GAL (3A9A; CNIO Monoclonal Antibodies Core Unit, AM(3A9A)) and GFP (Cell Signaling). For β-GAL and GFP double staining, the immunohistochemical reaction was developed using 3,30-diaminobenzidine tetrahydrochloride (DAB) (Chromomap DAB, Ventana, Roche) and purple chromogen (Discovery Purple Kit, Ventana, Roche), respectively. Nuclei were counterstained with Harrys’s hematoxylin. Pictures were taken using Olympus AX70 microscope.

Muñoz-Lorente M.A., Martínez P., Tejera Á., Whittemore K., Moisés-Silva A.C., Bosch F, & Blasco M.A. (2018). AAV9-mediated telomerase activation does not accelerate tumorigenesis in the context of oncogenic K-Ras-induced lung cancer. PLoS Genetics, 14(8), e1007562.

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Dual IHC Staining Protocol for AR and ACE2

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IHC was performed on 4-μm-thick formalin-fixed, paraffin-embedded (FFPE) tissue sections using anti-AR rabbit monoclonal primary antibody (prediluted, pH 9, catalog no. 760-4605, Roche-Ventana) and anti-ACE2 rabbit monoclonal primary antibody (1:100, pH 9, catalog no. GTX01160, GeneTex). IHC was carried out on the Benchmark XT automated slide staining system (Roche-Ventana Medical Systems) using the UltraView Universal diaminobenzidine (DAB) detection kit (catalog no. 760-500, Roche-Ventana) and Hematoxylin II (catalog no. 790-2208, Roche-Ventana) for counterstain. Dual IHC was performed consecutively, and signals were developed using the Universal DAB detection kit and the Discovery purple kit (catalog no. 760-229, Roche-Ventana). Staining was evaluated under 100× and 200× magnification using a bright-field microscope. See SI Appendix for scoring details.

Qiao Y., Wang X.M., Mannan R., Pitchiaya S., Zhang Y., Wotring J.W., Xiao L., Robinson D.R., Wu Y.M., Tien J.C., Cao X., Simko S.A., Apel I.J., Bawa P., Kregel S., Narayanan S.P., Raskind G., Ellison S.J., Parolia A., Zelenka-Wang S., McMurry L., Su F., Wang R., Cheng Y., Delekta A.D., Mei Z., Pretto C.D., Wang S., Mehra R., Sexton J.Z, & Chinnaiyan A.M. (2020). Targeting transcriptional regulation of SARS-CoV-2 entry factors ACE2 and TMPRSS2. Proceedings of the National Academy of Sciences of the United States of America, 118(1), e2021450118.

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Immunohistochemical Analysis of HtrA1 in Dry AMD

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Criteria for patient classification were absence of disorders of choroid and retina for the control patients (referred in the text as healthy subject) or a confirmed diagnosis of dry AMD by Eye care professional (refereed in the text as AMD patient). Sections of 4 μm thickness were obtained from formalin-fixed, paraffin-embedded human eye tissue from 5 healthy donors and 5 AMD patients. An immunohistochemestry (IHC) staining protocol was performed with the automated IHC research slide staining system Ventana Discovery Ultra. Stainings were carried out utilizing protocols and reagents according to the instructions of the manufacturer. An anti- HtrA1 (1:500) (Vierkotten et al., 2011 ) was employed as a primary antibody. A secondary antibody anti-rabbit HRP conjugated antibody (VENTANA, 760-4311) was used to detect the primary antibody. As chromogen, the Discovery Purple Kit (VENTANA, 760-229) was employed. Nuclear counterstaining was performed with hematoxylin.
An Olympus system VS120 slide scanner with a dry 40 × objective was employed to acquire the images of the stained retinas.

Melo E., Oertle P., Trepp C., Meistermann H., Burgoyne T., Sborgi L., Cabrera A.C., Chen C.Y., Hoflack J.C., Kam-Thong T., Schmucki R., Badi L., Flint N., Ghiani Z.E., Delobel F., Stucki C., Gromo G., Einhaus A., Hornsperger B., Golling S., Siebourg-Polster J., Gerber F., Bohrmann B., Futter C., Dunkley T., Hiller S., Schilling O., Enzmann V., Fauser S., Plodinec M, & Iacone R. (2017). HtrA1 Mediated Intracellular Effects on Tubulin Using a Polarized RPE Disease Model. EBioMedicine, 27, 258-274.

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In Situ Hybridization of TLINC Transcripts

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Specific in situ hybridization (ISH) riboprobes were designed to evaluate the expression of TLINC‐S and TLINC‐T transcripts (Supporting Table S1). Sense and antisense riboprobes were generated by in vitro transcription from PCR products incorporating the promoter of T7 RNA Polymerase. In vitro transcription was performed with 40 units T7 RNA Polymerase (Agilent) in the presence of 0.35 mM digoxygenin‐11‐UTP (Roche Diagnostics, Meylan, France). ISH was performed on the Discovery Automated IHC Stainer using the Ventana Detection Kit (Ventana Medical Systems, Tucson, AZ). Staining was performed using an anti‐digoxygenin‐horseradish peroxidase antibody (Roche Diagnostics), and signal detection was performed using a nitro‐blue tetrazolium/5‐bromo‐4‐chloro‐3′‐indolyphosphate or a Discovery purple kit (Ventana Medical Systems).

Merdrignac A., Angenard G., Allain C., Petitjean K., Bergeat D., Bellaud P., Fautrel A., Turlin B., Clément B., Dooley S., Sulpice L., Boudjema K, & Coulouarn C. (2018). A novel transforming growth factor beta‐induced long noncoding RNA promotes an inflammatory microenvironment in human intrahepatic cholangiocarcinoma. Hepatology Communications, 2(3), 254-269.

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Immunostaining of POWV and LGTV Proteins

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Anti-POWV and anti-LGTV virus immunoreactivities were detected using POWV ascites fluid (which detects multiple viral proteins; ATCC) (59 (link)) at a 1:2,000 dilution and anti-LGTV-E mouse monoclonal antibody (detects viral E protein; 11H12) at a 1:50 dilution, respectively. The secondary antibody was a ready-to-use Discovery OmniMap anti-mouse horseradish peroxidase conjugate (prediluted; Roche). The tissues were then processed for immunohistochemical staining using the Discovery Ultra automated processor (Ventana Medical Systems, Tucson, AZ) with a Discovery Purple kit (Ventana Medical Systems). Stained cross-sections of infected and mock-infected organs were viewed on an Olympus model BX51 microscrope, and images were collected using an Olympus model DP80 camera. Images were processed with Olympus cellSens Dimension v1.13 software. Images were captured at 20×, 40×, and 100×magnification (indicated in the figures) with LGTV or POWV protein staining denoted by a prominent purple color. Mock-infected organs were processed in parallel with infected organs at 96 hpi with antibody for LGTV and at 192 hpi with antibody for POWV in order to identify any nonspecific staining.

Grabowski J.M., Tsetsarkin K.A., Long D., Scott D.P., Rosenke R., Schwan T.G., Mlera L., Offerdahl D.K., Pletnev A.G, & Bloom M.E. (2017). Flavivirus Infection of Ixodes scapularis (Black-Legged Tick) Ex Vivo Organotypic Cultures and Applications for Disease Control. mBio, 8(4), e01255-17.

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Immunohistochemical Analysis of Tissue Samples

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Spleen, bone and kidney tissues were fixed in 4% (wt/vol) formaldehyde (Panreac) for 72 h and washed in 70% ethanol before paraffin embedding. Tissue sections were stained with H&E and with specific monoclonal antibodies (Supplementary Table 9). An automated immunostaining platform (Discovery XT-ULTRA, Ventana-Roche) was used. Briefly, sections stained with rat anti-CD138 (clone 281-2; 1:20,000 dilution) were incubated with rabbit anti-rat secondary antibody (BA4001; 1:100 dilution). Then, the sections were incubated with goat anti-rabbit-labeled polymer using the EnVision⁺ System (Dako), and peroxidase activity was revealed using DAB⁺ (Dako). For stains with monoclonal anti-c-MYC (Y69; 1:100 dilution) or anti-GFP (D5.1; 1:100 dilution), slides were incubated with the visualization systems (OmniMap anti-Rabbit) conjugated to horseradish peroxidase. Immunohistochemistry reactions were developed using 30-diaminobenzidine tetrahydrochloride (ChromoMap DAB, Ventana, Roche) and purple chromogen (Discovery Purple Kit, Ventana, Roche). Finally, nuclei were counterstained in Hematoxylin II. In selected BM samples, Giemsa or alkaline phosphatase staining was performed according to standard procedures.

Larrayoz M., Garcia-Barchino M.J., Celay J., Etxebeste A., Jimenez M., Perez C., Ordoñez R., Cobaleda C., Botta C., Fresquet V., Roa S., Goicoechea I., Maia C., Lasaga M., Chesi M., Bergsagel P.L., Larrayoz M.J., Calasanz M.J., Campos-Sanchez E., Martinez-Cano J., Panizo C., Rodriguez-Otero P., Vicent S., Roncador G., Gonzalez P., Takahashi S., Katz S.G., Walensky L.D., Ruppert S.M., Lasater E.A., Amann M., Lozano T., Llopiz D., Sarobe P., Lasarte J.J., Planell N., Gomez-Cabrero D., Kudryashova O., Kurilovich A., Revuelta M.V., Cerchietti L., Agirre X., San Miguel J., Paiva B., Prosper F, & Martinez-Climent J.A. (2023). Preclinical models for prediction of immunotherapy outcomes and immune evasion mechanisms in genetically heterogeneous multiple myeloma. Nature Medicine, 29(3), 632-645.

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