--- _id: '163' abstract: - lang: eng 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. article_processing_charge: No article_type: original author: - first_name: Siegfried full_name: Reipert, Siegfried last_name: Reipert - first_name: Helmuth full_name: Goldammer, Helmuth last_name: Goldammer - first_name: Christine full_name: Richardson, Christine last_name: Richardson - first_name: Martin full_name: Goldberg, Martin last_name: Goldberg - first_name: Timothy full_name: Hawkins, Timothy last_name: Hawkins - first_name: Elena full_name: Hollergschwandtner, Elena id: 3C054040-F248-11E8-B48F-1D18A9856A87 last_name: Hollergschwandtner - first_name: Walter full_name: Kaufmann, Walter id: 3F99E422-F248-11E8-B48F-1D18A9856A87 last_name: Kaufmann orcid: 0000-0001-9735-5315 - first_name: Sebastian full_name: Antreich, Sebastian last_name: Antreich - first_name: York full_name: Stierhof, York last_name: Stierhof citation: ama: 'Reipert S, Goldammer H, Richardson C, et al. Agitation modules: Flexible means to accelerate automated freeze substitution. Journal of Histochemistry and Cytochemistry. 2018;66(12):903-921. doi:10.1369/0022155418786698' 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. Journal of Histochemistry and Cytochemistry. SAGE Publications. https://doi.org/10.1369/0022155418786698' 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.” Journal of Histochemistry and Cytochemistry. SAGE Publications, 2018. https://doi.org/10.1369/0022155418786698.' ieee: 'S. Reipert et al., “Agitation modules: Flexible means to accelerate automated freeze substitution,” Journal of Histochemistry and Cytochemistry, vol. 66, no. 12. SAGE Publications, pp. 903–921, 2018.' 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.' mla: 'Reipert, Siegfried, et al. “Agitation Modules: Flexible Means to Accelerate Automated Freeze Substitution.” Journal of Histochemistry and Cytochemistry, vol. 66, no. 12, SAGE Publications, 2018, pp. 903–21, doi:10.1369/0022155418786698.' 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. date_created: 2018-12-11T11:44:57Z date_published: 2018-12-01T00:00:00Z date_updated: 2026-06-18T17:50:00Z day: '01' ddc: - '570' department: - _id: RySh - _id: EM-Fac doi: 10.1369/0022155418786698 external_id: isi: - '000452277700005' pmid: - '29969056' intvolume: ' 66' isi: 1 issue: '12' language: - iso: eng main_file_link: - open_access: '1' url: https://doi.org/10.1369/0022155418786698 month: '12' oa: 1 oa_version: Published Version page: 903-921 pmid: 1 publication: Journal of Histochemistry and Cytochemistry publication_identifier: issn: - 0022-1554 publication_status: published publisher: SAGE Publications quality_controlled: '1' scopus_import: '1' status: public title: 'Agitation modules: Flexible means to accelerate automated freeze substitution' type: journal_article user_id: 2DF688A6-F248-11E8-B48F-1D18A9856A87 volume: 66 year: '2018' ... --- OA_place: publisher OA_type: free access _id: '3931' abstract: - lang: eng 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. article_processing_charge: No article_type: original author: - first_name: Hui full_name: Zhang, Hui last_name: Zhang - first_name: Stephan full_name: Baader, Stephan last_name: Baader - first_name: Michael K full_name: Sixt, Michael K id: 41E9FBEA-F248-11E8-B48F-1D18A9856A87 last_name: Sixt orcid: 0000-0002-6620-9179 - first_name: Joachim full_name: Kappler, Joachim last_name: Kappler - first_name: Uwe full_name: Rauch, Uwe last_name: Rauch citation: 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. Journal of Histochemistry and Cytochemistry. 2004;52(7):915-922. doi:10.1369/jhc.3A6221.2004' apa: 'Zhang, H., Baader, S., Sixt, M. K., 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. Histochemical Society. https://doi.org/10.1369/jhc.3A6221.2004' 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.” Journal of Histochemistry and Cytochemistry. Histochemical Society, 2004. https://doi.org/10.1369/jhc.3A6221.2004.' 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,” Journal of Histochemistry and Cytochemistry, vol. 52, no. 7. Histochemical Society, pp. 915–922, 2004.' 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.' mla: 'Zhang, Hui, et al. “Neurocan-GFP Fusion Protein: A New Approach to Detect Hyaluronan on Tissue Sections and Living Cells.” Journal of Histochemistry and Cytochemistry, vol. 52, no. 7, Histochemical Society, 2004, pp. 915–22, doi:10.1369/jhc.3A6221.2004.' short: H. Zhang, S. Baader, M.K. Sixt, J. Kappler, U. Rauch, Journal of Histochemistry and Cytochemistry 52 (2004) 915–922. date_created: 2018-12-11T12:05:57Z date_published: 2004-07-01T00:00:00Z date_updated: 2026-09-01T11:22:42Z day: '01' doi: 10.1369/jhc.3A6221.2004 extern: '1' external_id: pmid: - '15208358' intvolume: ' 52' issue: '7' keyword: - hyaluronan - affinity histochemistry - neurocan - GFP - fusion protein - time-lapse video microscopy language: - iso: eng main_file_link: - open_access: '1' url: https://doi.org/10.1369/jhc.3A6221.2004 month: '07' oa: 1 oa_version: Published Version page: 915 - 922 pmid: 1 publication: Journal of Histochemistry and Cytochemistry publication_identifier: eissn: - 1551-5044 issn: - 0022-1554 publication_status: published publisher: Histochemical Society publist_id: '2196' quality_controlled: '1' scopus_import: '1' status: public title: 'Neurocan-GFP fusion protein: a new approach to detect hyaluronan on tissue sections and living cells' type: journal_article user_id: ba8df636-2132-11f1-aed0-ed93e2281fdd volume: 52 year: '2004' ... --- OA_type: closed access _id: '3929' abstract: - lang: eng 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. article_processing_charge: No article_type: original author: - first_name: Vanessa full_name: Witte, Vanessa last_name: Witte - first_name: Bernd full_name: Laffert, Bernd last_name: Laffert - first_name: Olaf full_name: Rosorius, Olaf last_name: Rosorius - first_name: Peter full_name: Lischka, Peter last_name: Lischka - first_name: Katja full_name: Blume, Katja last_name: Blume - first_name: Gunther full_name: Galler, Gunther last_name: Galler - first_name: Andrea full_name: Stilper, Andrea last_name: Stilper - first_name: Dieter full_name: Willbold, Dieter last_name: Willbold - first_name: Paola full_name: D'Aloja, Paola last_name: D'Aloja - first_name: Michael K full_name: Sixt, Michael K id: 41E9FBEA-F248-11E8-B48F-1D18A9856A87 last_name: Sixt orcid: 0000-0002-6620-9179 - first_name: Johanna full_name: Kolanus, Johanna last_name: Kolanus - first_name: Melanie full_name: Ott, Melanie last_name: Ott - first_name: Waldemar full_name: Kolanus, Waldemar last_name: Kolanus - first_name: Gerold full_name: Schuler, Gerold last_name: Schuler - first_name: Andreas full_name: Baur, Andreas last_name: Baur 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. Molecular Cell. 2004;13(2):179-190. doi:10.1016/S1097-2765(04)00004-8 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. Molecular Cell. Elsevier. https://doi.org/10.1016/S1097-2765(04)00004-8 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.” Molecular Cell. Elsevier, 2004. https://doi.org/10.1016/S1097-2765(04)00004-8. ieee: V. Witte et al., “HIV-1 Nef mimics an integrin receptor signal that recruits the polycomb group protein Eed to the plasma membrane,” Molecular Cell, vol. 13, no. 2. Elsevier, pp. 179–190, 2004. 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. mla: Witte, Vanessa, et al. “HIV-1 Nef Mimics an Integrin Receptor Signal That Recruits the Polycomb Group Protein Eed to the Plasma Membrane.” Molecular Cell, vol. 13, no. 2, Elsevier, 2004, pp. 179–90, doi:10.1016/S1097-2765(04)00004-8. 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. date_created: 2018-12-11T12:05:56Z date_published: 2004-01-30T00:00:00Z date_updated: 2026-09-01T11:49:45Z day: '30' doi: 10.1016/S1097-2765(04)00004-8 extern: '1' external_id: pmid: - '14759364 ' intvolume: ' 13' issue: '2' language: - iso: eng month: '01' oa_version: None page: 179 - 190 pmid: 1 publication: Molecular Cell publication_identifier: eissn: - 1551-5044 issn: - 0022-1554 publication_status: published publisher: Elsevier publist_id: '2197' quality_controlled: '1' scopus_import: '1' status: public title: HIV-1 Nef mimics an integrin receptor signal that recruits the polycomb group protein Eed to the plasma membrane type: journal_article user_id: ba8df636-2132-11f1-aed0-ed93e2281fdd volume: 13 year: '2004' ...