[{"title":"Agitation modules: Flexible means to accelerate automated freeze substitution","date_published":"2018-12-01T00:00:00Z","author":[{"full_name":"Reipert, Siegfried","last_name":"Reipert","first_name":"Siegfried"},{"first_name":"Helmuth","full_name":"Goldammer, Helmuth","last_name":"Goldammer"},{"first_name":"Christine","last_name":"Richardson","full_name":"Richardson, Christine"},{"full_name":"Goldberg, Martin","last_name":"Goldberg","first_name":"Martin"},{"last_name":"Hawkins","full_name":"Hawkins, Timothy","first_name":"Timothy"},{"first_name":"Elena","id":"3C054040-F248-11E8-B48F-1D18A9856A87","full_name":"Hollergschwandtner, Elena","last_name":"Hollergschwandtner"},{"full_name":"Kaufmann, Walter","orcid":"0000-0001-9735-5315","last_name":"Kaufmann","first_name":"Walter","id":"3F99E422-F248-11E8-B48F-1D18A9856A87"},{"last_name":"Antreich","full_name":"Antreich, Sebastian","first_name":"Sebastian"},{"full_name":"Stierhof, York","last_name":"Stierhof","first_name":"York"}],"publication_status":"published","page":"903-921","publication":"Journal of Histochemistry and Cytochemistry","quality_controlled":"1","pmid":1,"article_processing_charge":"No","citation":{"ama":"Reipert S, Goldammer H, Richardson C, et al. Agitation modules: Flexible means to accelerate automated freeze substitution. <i>Journal of Histochemistry and Cytochemistry</i>. 2018;66(12):903-921. doi:<a href=\"https://doi.org/10.1369/0022155418786698\">10.1369/0022155418786698</a>","apa":"Reipert, S., Goldammer, H., Richardson, C., Goldberg, M., Hawkins, T., Saeckl, E., … Stierhof, Y. (2018). Agitation modules: Flexible means to accelerate automated freeze substitution. <i>Journal of Histochemistry and Cytochemistry</i>. SAGE Publications. <a href=\"https://doi.org/10.1369/0022155418786698\">https://doi.org/10.1369/0022155418786698</a>","ista":"Reipert S, Goldammer H, Richardson C, Goldberg M, Hawkins T, Saeckl E, Kaufmann W, Antreich S, Stierhof Y. 2018. Agitation modules: Flexible means to accelerate automated freeze substitution. Journal of Histochemistry and Cytochemistry. 66(12), 903–921.","short":"S. Reipert, H. Goldammer, C. Richardson, M. Goldberg, T. Hawkins, E. Saeckl, W. Kaufmann, S. Antreich, Y. Stierhof, Journal of Histochemistry and Cytochemistry 66 (2018) 903–921.","mla":"Reipert, Siegfried, et al. “Agitation Modules: Flexible Means to Accelerate Automated Freeze Substitution.” <i>Journal of Histochemistry and Cytochemistry</i>, vol. 66, no. 12, SAGE Publications, 2018, pp. 903–21, doi:<a href=\"https://doi.org/10.1369/0022155418786698\">10.1369/0022155418786698</a>.","ieee":"S. Reipert <i>et al.</i>, “Agitation modules: Flexible means to accelerate automated freeze substitution,” <i>Journal of Histochemistry and Cytochemistry</i>, vol. 66, no. 12. SAGE Publications, pp. 903–921, 2018.","chicago":"Reipert, Siegfried, Helmuth Goldammer, Christine Richardson, Martin Goldberg, Timothy Hawkins, Elena Saeckl, Walter Kaufmann, Sebastian Antreich, and York Stierhof. “Agitation Modules: Flexible Means to Accelerate Automated Freeze Substitution.” <i>Journal of Histochemistry and Cytochemistry</i>. SAGE Publications, 2018. <a href=\"https://doi.org/10.1369/0022155418786698\">https://doi.org/10.1369/0022155418786698</a>."},"type":"journal_article","publication_identifier":{"issn":["0022-1554"]},"_id":"163","status":"public","month":"12","volume":66,"abstract":[{"text":"For ultrafast fixation of biological samples to avoid artifacts, high-pressure freezing (HPF) followed by freeze substitution (FS) is preferred over chemical fixation at room temperature. After HPF, samples are maintained at low temperature during dehydration and fixation, while avoiding damaging recrystallization. This is a notoriously slow process. McDonald and Webb demonstrated, in 2011, that sample agitation during FS dramatically reduces the necessary time. Then, in 2015, we (H.G. and S.R.) introduced an agitation module into the cryochamber of an automated FS unit and demonstrated that the preparation of algae could be shortened from days to a couple of hours. We argued that variability in the processing, reproducibility, and safety issues are better addressed using automated FS units. For dissemination, we started low-cost manufacturing of agitation modules for two of the most widely used FS units, the Automatic Freeze Substitution Systems, AFS(1) and AFS2, from Leica Microsystems, using three dimensional (3D)-printing of the major components. To test them, several labs independently used the modules on a wide variety of specimens that had previously been processed by manual agitation, or without agitation. We demonstrate that automated processing with sample agitation saves time, increases flexibility with respect to sample requirements and protocols, and produces data of at least as good quality as other approaches.","lang":"eng"}],"date_updated":"2026-06-18T17:50:00Z","issue":"12","article_type":"original","isi":1,"oa_version":"Published Version","oa":1,"date_created":"2018-12-11T11:44:57Z","user_id":"2DF688A6-F248-11E8-B48F-1D18A9856A87","language":[{"iso":"eng"}],"external_id":{"pmid":["29969056"],"isi":["000452277700005"]},"department":[{"_id":"RySh"},{"_id":"EM-Fac"}],"intvolume":"        66","year":"2018","main_file_link":[{"open_access":"1","url":"https://doi.org/10.1369/0022155418786698"}],"publisher":"SAGE Publications","doi":"10.1369/0022155418786698","day":"01","scopus_import":"1","ddc":["570"]},{"date_created":"2018-12-11T12:05:57Z","user_id":"ba8df636-2132-11f1-aed0-ed93e2281fdd","oa":1,"language":[{"iso":"eng"}],"external_id":{"pmid":["15208358"]},"OA_place":"publisher","intvolume":"        52","year":"2004","main_file_link":[{"open_access":"1","url":"https://doi.org/10.1369/jhc.3A6221.2004"}],"doi":"10.1369/jhc.3A6221.2004","publisher":"Histochemical Society","OA_type":"free access","day":"01","publist_id":"2196","scopus_import":"1","title":"Neurocan-GFP fusion protein: a new approach to detect hyaluronan on tissue sections and living cells","date_published":"2004-07-01T00:00:00Z","author":[{"first_name":"Hui","last_name":"Zhang","full_name":"Zhang, Hui"},{"full_name":"Baader, Stephan","last_name":"Baader","first_name":"Stephan"},{"first_name":"Michael K","id":"41E9FBEA-F248-11E8-B48F-1D18A9856A87","full_name":"Sixt, Michael K","orcid":"0000-0002-6620-9179","last_name":"Sixt"},{"first_name":"Joachim","last_name":"Kappler","full_name":"Kappler, Joachim"},{"first_name":"Uwe","full_name":"Rauch, Uwe","last_name":"Rauch"}],"page":"915 - 922","publication_status":"published","quality_controlled":"1","pmid":1,"publication":"Journal of Histochemistry and Cytochemistry","extern":"1","type":"journal_article","publication_identifier":{"eissn":["1551-5044"],"issn":["0022-1554"]},"article_processing_charge":"No","citation":{"apa":"Zhang, H., Baader, S., Sixt, M. K., Kappler, J., &#38; Rauch, U. (2004). Neurocan-GFP fusion protein: a new approach to detect hyaluronan on tissue sections and living cells. <i>Journal of Histochemistry and Cytochemistry</i>. Histochemical Society. <a href=\"https://doi.org/10.1369/jhc.3A6221.2004\">https://doi.org/10.1369/jhc.3A6221.2004</a>","ama":"Zhang H, Baader S, Sixt MK, Kappler J, Rauch U. Neurocan-GFP fusion protein: a new approach to detect hyaluronan on tissue sections and living cells. <i>Journal of Histochemistry and Cytochemistry</i>. 2004;52(7):915-922. doi:<a href=\"https://doi.org/10.1369/jhc.3A6221.2004\">10.1369/jhc.3A6221.2004</a>","chicago":"Zhang, Hui, Stephan Baader, Michael K Sixt, Joachim Kappler, and Uwe Rauch. “Neurocan-GFP Fusion Protein: A New Approach to Detect Hyaluronan on Tissue Sections and Living Cells.” <i>Journal of Histochemistry and Cytochemistry</i>. Histochemical Society, 2004. <a href=\"https://doi.org/10.1369/jhc.3A6221.2004\">https://doi.org/10.1369/jhc.3A6221.2004</a>.","ieee":"H. Zhang, S. Baader, M. K. Sixt, J. Kappler, and U. Rauch, “Neurocan-GFP fusion protein: a new approach to detect hyaluronan on tissue sections and living cells,” <i>Journal of Histochemistry and Cytochemistry</i>, vol. 52, no. 7. Histochemical Society, pp. 915–922, 2004.","mla":"Zhang, Hui, et al. “Neurocan-GFP Fusion Protein: A New Approach to Detect Hyaluronan on Tissue Sections and Living Cells.” <i>Journal of Histochemistry and Cytochemistry</i>, vol. 52, no. 7, Histochemical Society, 2004, pp. 915–22, doi:<a href=\"https://doi.org/10.1369/jhc.3A6221.2004\">10.1369/jhc.3A6221.2004</a>.","short":"H. Zhang, S. Baader, M.K. Sixt, J. Kappler, U. Rauch, Journal of Histochemistry and Cytochemistry 52 (2004) 915–922.","ista":"Zhang H, Baader S, Sixt MK, Kappler J, Rauch U. 2004. Neurocan-GFP fusion protein: a new approach to detect hyaluronan on tissue sections and living cells. Journal of Histochemistry and Cytochemistry. 52(7), 915–922."},"_id":"3931","status":"public","keyword":["hyaluronan","affinity histochemistry","neurocan","GFP","fusion protein","time-lapse video microscopy"],"article_type":"original","abstract":[{"text":"Hyaluronan is an unsulfated glycosaminoglycan (GAG) that is ubiquitously expressed in the extracellular matrix (ECM) of all vertebrates, where hyaluronan rich matrices constitute a particular permissive environment for the development of complex biological structures and also for tumor progression. Because of its conserved structure and ubiquitous expression, antibodies for its histochemical detection cannot be produced. We have engineered a fusion protein, neurocan-GFP, and expressed it as a secreted molecule in mammalian cells. Neurocan-GFP fusion protein specifically binds to hyaluronan and directly visualizes hyaluronan on tissue sections, revealing a very detailed picture of hyaluronan distribution. The fluorescent fusion protein can be used in combination with antibodies and nuclear markers for double or triple staining. In addition, it is suitable to visualize hyaluronan on living cells by time-lapse video microscopy. The successful production and application of the neurocan-GFP fusion protein opens up new perspectives for using GFP fusion proteins as detection tools in histological and cytological studies complementing conventional antibody and biotin/avidin techniques.","lang":"eng"}],"volume":52,"month":"07","date_updated":"2026-09-01T11:22:42Z","issue":"7","oa_version":"Published Version"},{"author":[{"last_name":"Witte","full_name":"Witte, Vanessa","first_name":"Vanessa"},{"first_name":"Bernd","last_name":"Laffert","full_name":"Laffert, Bernd"},{"first_name":"Olaf","full_name":"Rosorius, Olaf","last_name":"Rosorius"},{"last_name":"Lischka","full_name":"Lischka, Peter","first_name":"Peter"},{"first_name":"Katja","last_name":"Blume","full_name":"Blume, Katja"},{"full_name":"Galler, Gunther","last_name":"Galler","first_name":"Gunther"},{"full_name":"Stilper, Andrea","last_name":"Stilper","first_name":"Andrea"},{"full_name":"Willbold, Dieter","last_name":"Willbold","first_name":"Dieter"},{"last_name":"D'Aloja","full_name":"D'Aloja, Paola","first_name":"Paola"},{"id":"41E9FBEA-F248-11E8-B48F-1D18A9856A87","first_name":"Michael K","last_name":"Sixt","orcid":"0000-0002-6620-9179","full_name":"Sixt, Michael K"},{"last_name":"Kolanus","full_name":"Kolanus, Johanna","first_name":"Johanna"},{"first_name":"Melanie","full_name":"Ott, Melanie","last_name":"Ott"},{"full_name":"Kolanus, Waldemar","last_name":"Kolanus","first_name":"Waldemar"},{"first_name":"Gerold","full_name":"Schuler, Gerold","last_name":"Schuler"},{"full_name":"Baur, Andreas","last_name":"Baur","first_name":"Andreas"}],"date_published":"2004-01-30T00:00:00Z","title":"HIV-1 Nef mimics an integrin receptor signal that recruits the polycomb group protein Eed to the plasma membrane","pmid":1,"quality_controlled":"1","publication":"Molecular Cell","extern":"1","publication_status":"published","page":"179 - 190","_id":"3929","status":"public","publication_identifier":{"issn":["0022-1554"],"eissn":["1551-5044"]},"type":"journal_article","citation":{"ama":"Witte V, Laffert B, Rosorius O, et al. HIV-1 Nef mimics an integrin receptor signal that recruits the polycomb group protein Eed to the plasma membrane. <i>Molecular Cell</i>. 2004;13(2):179-190. doi:<a href=\"https://doi.org/10.1016/S1097-2765(04)00004-8\">10.1016/S1097-2765(04)00004-8</a>","apa":"Witte, V., Laffert, B., Rosorius, O., Lischka, P., Blume, K., Galler, G., … Baur, A. (2004). HIV-1 Nef mimics an integrin receptor signal that recruits the polycomb group protein Eed to the plasma membrane. <i>Molecular Cell</i>. Elsevier. <a href=\"https://doi.org/10.1016/S1097-2765(04)00004-8\">https://doi.org/10.1016/S1097-2765(04)00004-8</a>","short":"V. Witte, B. Laffert, O. Rosorius, P. Lischka, K. Blume, G. Galler, A. Stilper, D. Willbold, P. D’Aloja, M.K. Sixt, J. Kolanus, M. Ott, W. Kolanus, G. Schuler, A. Baur, Molecular Cell 13 (2004) 179–190.","ista":"Witte V, Laffert B, Rosorius O, Lischka P, Blume K, Galler G, Stilper A, Willbold D, D’Aloja P, Sixt MK, Kolanus J, Ott M, Kolanus W, Schuler G, Baur A. 2004. HIV-1 Nef mimics an integrin receptor signal that recruits the polycomb group protein Eed to the plasma membrane. Molecular Cell. 13(2), 179–190.","ieee":"V. Witte <i>et al.</i>, “HIV-1 Nef mimics an integrin receptor signal that recruits the polycomb group protein Eed to the plasma membrane,” <i>Molecular Cell</i>, vol. 13, no. 2. Elsevier, pp. 179–190, 2004.","chicago":"Witte, Vanessa, Bernd Laffert, Olaf Rosorius, Peter Lischka, Katja Blume, Gunther Galler, Andrea Stilper, et al. “HIV-1 Nef Mimics an Integrin Receptor Signal That Recruits the Polycomb Group Protein Eed to the Plasma Membrane.” <i>Molecular Cell</i>. Elsevier, 2004. <a href=\"https://doi.org/10.1016/S1097-2765(04)00004-8\">https://doi.org/10.1016/S1097-2765(04)00004-8</a>.","mla":"Witte, Vanessa, et al. “HIV-1 Nef Mimics an Integrin Receptor Signal That Recruits the Polycomb Group Protein Eed to the Plasma Membrane.” <i>Molecular Cell</i>, vol. 13, no. 2, Elsevier, 2004, pp. 179–90, doi:<a href=\"https://doi.org/10.1016/S1097-2765(04)00004-8\">10.1016/S1097-2765(04)00004-8</a>."},"article_processing_charge":"No","oa_version":"None","article_type":"original","issue":"2","date_updated":"2026-09-01T11:49:45Z","volume":13,"month":"01","abstract":[{"text":"The Nef protein of human and simian immunodeficiency virus (HIV/SIV) is believed to interfere with T cell activation signals by forming a signaling complex at the plasma membrane. Composition and function of the complex are not fully understood. Here we report that Nef recruits the Polycomb Group (PcG) protein Eed, so far known as a nuclear factor and repressor of transcription, to the membrane of cells. The Nef-induced translocation of Eed led to a potent stimulation of Tat-dependent HIV transcription, implying that Eed removal from the nucleus is required for optimal Tat function. Similar to Nef action, activation of integrin receptors recruited Eed to the plasma membrane, also leading to enhanced Tat/Nef-mediated transcription. Our results suggest a link between membrane-associated activation processes and transcriptional derepression and demonstrate how HIV exploits this mechanism.","lang":"eng"}],"external_id":{"pmid":["14759364 "]},"language":[{"iso":"eng"}],"user_id":"ba8df636-2132-11f1-aed0-ed93e2281fdd","date_created":"2018-12-11T12:05:56Z","year":"2004","intvolume":"        13","day":"30","OA_type":"closed access","doi":"10.1016/S1097-2765(04)00004-8","publisher":"Elsevier","scopus_import":"1","publist_id":"2197"}]