Enter your email address below and we will send you your username, If the address matches an existing account you will receive an email with instructions to retrieve your username. Contact Us | Product Reviews », Relevant content specific to your research interests. This Open Access article is distributed under the terms of the Creative Commons Attribution Noncommercial License ( ClearLight Biotechnologies, Inc. All Rights Reserved. As a result, when the tissue is completely infiltrated with the clearing agent, it becomes translucent. We observe some errors during monitoring, resulting from tissue slippage that occurs when tissue shrinks as a result of dehydration or clearing, or during reagent transitions as fluid drains from the cassette. While the CLARITY protocol is published for any lab to perform on its own, to achieve the best results for clearing, antibody penetration, and imaging we encourage researchers to leverage our expertise and intellectual property at ClearLight Biotechnologies. For the kidney tissue, the average TOF trace (Figure 3b) and the τ90 for 70% ethanol was 3.71 hours, 90% ethanol was 1.59 hours, 100% ethanol was 1.40, and clearing in xylene was 1.27 hours. We monitored incomplete dehydration of tissue by skipping a key processing step, dehydration in absolute ethanol, and then correlated the τ90 with poor histomorphology, demonstrating that the technique can detect significant processing errors. iDISCO and Visikol® HISTO™ samples appear to have an overall reduction in the PAX8 signal while the DAPI signal is strong. We did not choose to monitor paraffin wax infiltration in this study, largely because the geometry of our prototype TOF device complicated removal of residual hardened wax from the instrument. The time constant τ90 for the 70% ethanol incubation was 2.92 and R2 of 0.998 for the 70% ethanol step and 1.48 ± 0.16 hours and R2 of 0.999 for the 90% ethanol dehydration step (R2 values in these experiments correspond to a fit of the actual TOF trace to a single exponential decay). 4a). There are many potential directions for our next steps with this research. A system for rapid diagnostic tissue preparation. Submit News Tips | (a) Properly processed normal kidney TOF trace through each reagent for a specific piece of tissue. We applaud the research community and their involvement in the drug discovery and development process. This may render a tissue “clear” but does it actually preserve the antigen and receptors for the given targets of interest in the tissue? It is possible to use these data to determine complete diffusion of reagents irrespective of tissue size. Tag: What is clearing in tissue processing? (d) Liver tissue TOF trace. The CUBIC sample appears slightly hazy, and although the iDISCO and Visikol® HISTO™ samples appear very clear to the naked eye, they also look more handled and slightly degraded. Please read our policies on privacy, shipping and returns. The time needed for reagent diffusion in the dehydration and clearing steps of tissue processing is shown as an average for all tissue types (Fig. 3). are employees of Ventana Medical Systems, Inc. No competing financial interests exist for the remaining authors. Xylene: It is the most commonly used clearing agent in histopathology laboratory. Slides were deparaffinized using EZPrep (Ventana Medical Systems, Inc.) at 90°C. YouTube | [Ultrasound-mediated treatment of pathology archival samples]. The prepared tissues were derived from the same tissue sample. In this study, we have demonstrated the ability of the TOF ultrasound technology to monitor the complete tissue processing cycle by monitoring diffusion of processing reagents in 270 tissues from 13 different tissue. Ultrasound has been used experimentally for degradation of DNA. We omitted the 100% ethanol incubation in the processing protocol, and instead directly moved tissue from 90% ethanol into xylene so that residual water would be left in the tissue. Time constants τ63 and τ90 for alcohols and xylene by tissue type for complete TPP and rapid TPP. -, Xie R, Chung J-Y, Ylaya K, et al. Hofman V, Ilie M, Gavric-Tanga V, Lespinet V, Mari M, Lassalle S, Butori C, Coelle C, Bordone O, Selva E, Lamy A, Sabourin JC, Hofman P. Ann Pathol. Ultrasound time-of-flight (TOF) technology has been successfully used to monitor the critical processing step of tissue fixation with formalin. This MCF-7 mouse xenograft was immunostained using Ki67. We collected 270 tissues from 13 different tissue types (Table 2) and monitored the diffusion time using TOF. The resulting protocol yields tissue that is ready for histologic sectioning 18 hours after excision for a variety of tissue types. A Review of preanalytical factors affecting molecular, protein, and morphological analysis of formalin-fixed, paraffin-embedded (FFPE) tissue: How well do you know your FFPE specimen?