Pt link platform

Manufactured by Agilent Technologies

Sourced in Denmark

The PT link platform is a laboratory equipment product from Agilent Technologies. It serves as a connectivity solution, enabling the integration of various analytical instruments and devices within a lab environment. The core function of the PT link platform is to facilitate seamless data exchange and instrument control across the lab.

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5 protocols using pt link platform

Tissue Immunohistochemistry with DAB Detection

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Tissue was fixed with 4% formaldehyde in PBS for 48h and sent to VAI histology core for embedding. Deparaffinization and antigen retrieval were performed on the Dako PT link platform using Dako High pH retriever buffer for 20 minutes at 97°C. Staining was performed utilizing Dako Autostainer Link 48, and Dako Rabbit Polymer HRP as secondary antibody for 20 minutes following primary antibody incubation for 30 minutes. DAB detection was performed using Dako EnVision Flex Chromagen for 10 minutes and Dako Flex Hematoxylin for 5 minutes. Aperio scanning of slides was performed utilizing Leica Aperio AT2 system.

Avequin T., Lau K.H., Waldhart A.N., Guak H., Dykstra H., Krawczyk C, & Wu N. (2023). Differential effects of sugar and fat on adipose tissue inflammation. iScience, 26(7), 107163.

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Immunohistochemical Detection of AMH

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Slides from TMAs (4 μm-thick) were used for immunohistochemistry (IHC) reactions, which were performed using DakoAutostainer Link48 (Dako, Glostrup, Denmark). In order to deparaffinize, rehydrate and unmask the antigens the sections were boiled in EnVision FLEX Target Retrieval Solution (pH 9, 20 min, 97 °C; Dako) using the PTLink platform (Dako, Glostrup, Denmark). Afterwards, slides were incubated for 5 min with Envision Flex Peroxidase-Blocking Reagent (Dako, Glostrup, Denmark) to block endogenous peroxidase. As primary antibodies (20 min, RT), rabbit polyclonal antibodies against AMH (1:100, ab84952, Abcam, Cambridge, UK) were used. Next, slides were incubated with EnVision FLEX/HRP (20 min, RT), and the reaction was visualized (10 min, RT) with freshly prepared 3,3′-diaminobenzidine (DAB). Additionally, slides were counterstained for 5 min with EnVision FLEX Hematoxylin (Dako, Glostrup, Denmark). Finally, slides were dehydrated in ethanol (70%, 96%, absolute) and xylene, then mounted with Dako Mounting Medium (Dako, Glostrup, Denmark). Slides were evaluated using the Olympus BX41 light microscope (Olympus, Japan). Control tissues included the human prostate.

Gowkielewicz M., Lipka A., Piotrowska A., Szadurska-Noga M., Nowakowski J.J., Dzięgiel P., Majewski M.K., Jozwik M, & Majewska M. (2019). Anti-Müllerian Hormone Expression in Endometrial Cancer Tissue. International Journal of Molecular Sciences, 20(6), 1325.

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Immunohistochemical Staining Protocol

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Waldhart A.N., Lau K.H., Dykstra H., Avequin T, & Wu N. (2023). Optimal HSF1 activation in response to acute cold stress in BAT requires nuclear TXNIP. iScience, 26(5), 106538.

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Immunohistochemical Quantification of BAT

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Tissue was fixed with 4% formaldehyde in PBS for 48h and sent to VAI histology core for embedding. Deparaffinization and antigen retrieval performed on Dako PT link platform using Dako High pH retriever buffer for 20 minutes at 97 degrees C. Staining performed utilizing Dako Autostainer Link 48, utilizing Dako Rabbit Polymer HRP for secondary for 20 minutes following primary antibody incubation for 30 minutes. DAB detection performed using Dako EnVision Flex Chromagen for 10 minutes and Dako Flex Hematoxylin for 5 minutes. Aperio scanning of slides was performed utilizing Leica Aperio AT2 system.
ImageJ software (NIH, Bethesda, MD) was used to quantitate the number of nuclei that were stained in each experimental condition. Positively stained nuclei were divided by the total number of nuclei to yield the percent of positive nuclei in BAT.

Lau K.H., Waldhart A.N., Dykstra H., Avequin T, & Wu N. (2022). PPARγ and C/EBPα response to acute cold stress in brown adipose tissue. iScience, 26(1), 105848.

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Immunoblotting and IHC Experiments

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Immunoblotting and IHC experiments were performed as described before (14 (link)). Gels shown are representative of at least three independent experiments. For immunoblotting, proteins were extracted using RIPA buffer, and primary antibodies were incubated overnight. For IHC, 5-μm formalin-fixed, paraffin-embedded (FFPE) sections underwent epitope retrieval using the Dako PT Link platform. Staining was on the Dako Link 48 autostainer, with amplification using EnVision FLEX mouse linkers, and visualization using the EnVision FLEX high-sensitivity visualization system (Dako). For WLS, sections were stained with an anti-WLS antibody YJ5 (Biolegand; 1:200). The mCRPC TMA in figure 4 contains 20 cases from the University of Washington rapid autopsy program. PORCN staining was similarly performed with anti-PORCN antibody (ab105543, Abcam, 1:200, RRID:AB_10860951). Primary antibodies are detailed in Supplemental Materials.

Ma F., Arai S., Wang K., Calagua C., Yuan A.R., Poluben L., Gu Z., Russo J.W., Einstein D.J., Ye H., He M.X., Liu Y., Van Allen E., Sowalsky A.G., Bhasin M.K., Yuan X, & Balk S.P. (2022). Autocrine canonical Wnt signaling primes noncanonical signaling through ROR1 in metastatic castration-resistant prostate cancer. Cancer research, 82(8), 1518-1533.

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