{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2025,9,26]],"date-time":"2025-09-26T09:10:12Z","timestamp":1758877812206,"version":"3.44.0"},"reference-count":27,"publisher":"MDPI AG","issue":"18","license":[{"start":{"date-parts":[[2025,9,18]],"date-time":"2025-09-18T00:00:00Z","timestamp":1758153600000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100001659","name":"German Research Foundation","doi-asserted-by":"crossref","award":["INST 152\/876-1 FUGG"],"award-info":[{"award-number":["INST 152\/876-1 FUGG"]}],"id":[{"id":"10.13039\/501100001659","id-type":"DOI","asserted-by":"crossref"}]},{"name":"Open Access Publication Fund of UKE\u2014Universit\u00e4tsklinikum Hamburg-Eppendorf"}],"content-domain":{"domain":["www.mdpi.com"],"crossmark-restriction":true},"short-container-title":["Cells"],"abstract":"<jats:p>Background: The disialoganglioside GD2, located at the plasma membrane, is selectively overexpressed in various solid tumors, where it contributes to tumor growth and the development of an aggressive tumor phenotype. Thus, over the last two decades GD2 has been gaining importance both as a tumor marker and a therapy target. In neuroblastoma, anti-GD2 monoclonal antibodies and CAR T-cells have become an integral part of the multimodal treatment for relapsed or refractory high-risk cases, which continue to associate with poor prognosis. GD2 characterization in neuroblastoma is well established for bone marrow aspirates and biopsies, but remains challenging in tumoral tissue samples, mostly due to epitope loss upon fixation. Aims: The aim of our work was to assess a new protocol by staining GD2 in tissue specimens prior to fixation. Methods: Positive controls were tissue specimens from patients with histologically confirmed neuroblastoma and GD2 expression in bone marrow aspirate (n = 5). Nephroblastoma or Hodgkin lymphoma samples were considered as negative controls (n = 5). Tissue staining was performed prior to fixation with either anti-GD2 antibody or isotype control, followed by secondary antibody staining and subsequent paraffinization. To examine GD2 staining before and after paraffinization, fluorescence images were acquired using 3D and 2D immunofluorescence microscopy techniques respectively. Results: GD2 signal was detected in all positive controls, while absent in all negative controls. After fixation, paraffinization and slicing no relevant signal loss was observed. Nevertheless, sufficient staining of 3D specimens required long incubation times, which led to increased cytolysis of the unfixed tissue. Conclusions: We were able to establish and validate a novel protocol to reliably perform immunostaining of the membrane antigen GD2 in unfixed, primary neuroblastoma tissue. Although including few limitations, this staining workflow enables relatively quick assessment of GD2 status and thus, might represent a relevant diagnostic tool within the framework of tumor staging and precision medicine.<\/jats:p>","DOI":"10.3390\/cells14181462","type":"journal-article","created":{"date-parts":[[2025,9,18]],"date-time":"2025-09-18T12:24:25Z","timestamp":1758198265000},"page":"1462","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["A Backwards Approach to GD2 Immunofluorescence in Human Neuroblastoma Tissue Samples: From Staining to Slicing"],"prefix":"10.3390","volume":"14","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-9703-6434","authenticated-orcid":false,"given":"Sara","family":"Peggion","sequence":"first","affiliation":[{"name":"Department of Pediatric Surgery, University Medical Centre Hamburg-Eppendorf, Martinistrasse 52, 20246 Hamburg, Germany"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Clara","family":"Volz","sequence":"additional","affiliation":[{"name":"Department of Pediatric Surgery, University Medical Centre Hamburg-Eppendorf, Martinistrasse 52, 20246 Hamburg, Germany"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Magdalena","family":"Trochimiuk","sequence":"additional","affiliation":[{"name":"Department of Pediatric Surgery, University Medical Centre Hamburg-Eppendorf, Martinistrasse 52, 20246 Hamburg, Germany"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-8220-7002","authenticated-orcid":false,"given":"Isabelle Ariane","family":"Bley","sequence":"additional","affiliation":[{"name":"Division of Pediatric Stem Cell Transplantation and Immunology, Clinic of Pediatric Hematology and Oncology, University Medical Center Hamburg-Eppendorf, 20246 Hamburg, Germany"},{"name":"Research Institute Children\u2019s Cancer Center Hamburg, 20251 Hamburg, Germany"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"J\u00falia","family":"Ramos","sequence":"additional","affiliation":[{"name":"Institut Bonanova, Circumval.laci\u00f3 8, 08003 Barcelona, Spain"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-7142-8758","authenticated-orcid":false,"given":"Konrad","family":"Reinshagen","sequence":"additional","affiliation":[{"name":"Department of Pediatric Surgery, University Medical Centre Hamburg-Eppendorf, Martinistrasse 52, 20246 Hamburg, Germany"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Laia","family":"Pagerols Raluy","sequence":"additional","affiliation":[{"name":"Department of Pediatric Surgery, University Medical Centre Hamburg-Eppendorf, Martinistrasse 52, 20246 Hamburg, Germany"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1968","published-online":{"date-parts":[[2025,9,18]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","unstructured":"Machy, P., Mortier, E., and Birkl\u00e9, S. 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