PMC:4996397 / 5870-9183
Annnotations
{"target":"https://pubannotation.org/docs/sourcedb/PMC/sourceid/4996397","sourcedb":"PMC","sourceid":"4996397","source_url":"https://www.ncbi.nlm.nih.gov/pmc/4996397","text":"2. Experimental Section: Study Design\nFor the purpose of this review we created several TMAs under defined conditions. Two of them (Figure 1B,D) were built by means of the TMA-GM instrument. Moreover, we took an old tissue array (Figure 1C) prepared ten years ago with the original TMA machine developed in 1998 by Beecher et al. and modified by Mirlacher [25] and another array (Figure 1A) constructed in 2013 with a TMA-M device. In addition, we selected donor blocks representative for various organs and histopathological characteristics. \nArray layout was defined indicating the number and location of tissue core to be collected from each donor block, their positions on the array to be constructed (recipient block), the diameter of each carrot and the distance between each spot on the recipient block. Several tissues were selected from the donor TMAs to be re-punched and transferred to the new TMA. Green and red circles indicate the selected tissues to be transferred (Figure 1) and their positions on the newly created TMAs (Figure 2). \nOnce the new TMA was built, the block was extracted from the TMA-GM and put on a glass slide. With the thumb and trigger finger the TMA block was pressed to the glass slide. The block up-side-down on the glass slide was incubated overnight at 42 °C. In the morning, after the TMA block was left to cool at room temperature (RT) for approximately 30 minutes and the glass slide removed, and the block was ready to be used. \nA section of each donor block was cut using a standard microtome. The slides were stained with Haematoxylin and Eosin (H\u0026E) and scanned. The pictures were checked by the pathologist on the computer screen by means of the Panoramic Viewer® (3D-Histech, Sysmex AG) program. The designed layout was loaded on TMA-GM. Images of donor blocks and corresponding histological slides were overlaid using the TMA-GM software. The designated tissue spots on the donor TMAs were directly selected according to the respective array coordinates (Figure 1 and Figure 2). Further slides of the donor blocks, as well as of the newly created TMA blocks with transferred tissues were stained with five antibodies: CD34 (Ventana 790-2927), CD45 (Ventana 760-4279), Smooth Muscle Actin (1A4; Ventana 760-2833), pan-Keratin (Ventana 760-2595) and Ki67 (Dako IR626). All stainings were performed with the BenchMark XT (Ventana Medical System Inc, Tucson, AZ, USA).\nFigure 1 Schematic overview of TMA donor blocks. (A) TMA performed by TMA-M (core diameter is 1 mm, and distance between cores is 0.5 mm). (B) TMA performed by TMA-GM (core diameter is 1 mm, and distance between cores is 0.7 mm). (C) TMA performed by old machine (core diameter is 0.6 mm, and distance between cores is 0.6 mm). (D) TMA performed with TMA-GM (core diameter is 0.6 mm, and distance between cores is 0.6 mm). Red and Green circles represent the selected tissues in each TMA donor block to be re-punched and transferred to a new recipient block. (For donor TMAs with 1mm diameter tissues, red circles represent tissues punched with TMA-M (A), and green circles represent tissues punched with TMA-GM (B). For donor TMAs with 0.6 mm diameter tissues, red circles represent tissues punched with original TMA instrument (C), and green circles represent tissues punched with TMA-GM (D).\n\n3","divisions":[{"label":"Title","span":{"begin":0,"end":37}},{"label":"Figure caption","span":{"begin":2415,"end":3312}}],"tracks":[{"project":"2_test","denotations":[{"id":"27600223-15205686-69477664","span":{"begin":357,"end":359},"obj":"15205686"}],"attributes":[{"subj":"27600223-15205686-69477664","pred":"source","obj":"2_test"}]}],"config":{"attribute types":[{"pred":"source","value type":"selection","values":[{"id":"2_test","color":"#da93ec","default":true}]}]}}