Journal of Neuro-Oncology

, Volume 135, Issue 1, pp 21–28 | Cite as

Frozen tissue preparation for high-resolution multiplex histological analyses of human brain specimens

Laboratory Investigation


The availability of a comprehensive tissue library is essential for elucidating the function and pathology of human brains. Considering the irreplaceable status of the formalin-fixation-paraffin-embedding (FFPE) preparation in routine pathology and the advantage of ultra-low temperature to preserve nucleic acids and proteins for multi-omics studies, these methods have become major modalities for the construction of brain tissue libraries. Nevertheless, the use of FFPE and snap-frozen samples is limited in high-resolution histological analyses because the preparation destroys tissue integrity and/or many important cellular markers. To overcome these limitations, we detailed a protocol to prepare and analyze frozen human brain samples that is particularly suitable for high-resolution multiplex immunohistological studies. As an alternative, we offered an optimized procedure to rescue snap-frozen tissues for the same purpose. Importantly, we provided a guideline to construct libraries of frozen tissue with minimal effort, cost and space. Taking advantage of this new tissue preparation modality to nicely preserve the cellular information that was otherwise damaged using conventional methods and to effectively remove tissue autofluorescence, we described the high-resolution landscape of the cellular composition in both lower-grade gliomas and glioblastoma multiforme samples. Our work showcases the great value of fixed frozen tissue in understanding the cellular mechanisms of CNS functions and abnormalities.


Frozen glioma tissue Oligodendrocyte precursor cell (OPC) Multiplex immunofluorescence staining Brain tissue library Autofluorescence. 



We appreciate the technique support from the Human Brain Bank of Zhejiang University. This work is supported by the National Key Research and Development Program of China, Stem Cell and Translational Research (2016YFA0101201 to C.L.), the Science Foundation for Distinguished Young Scientists of Zhejiang Province (LR17H160001 to C.L.), the National Science Foundation of China (81673035 to C.L.), the Thousand Talent Program for Young Outstanding Scientists, China (to C.L.) and Science funding of Zhejiang Province (LY17H160016 to H.S.).

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflicts of interest.

Informed consent

Informed consent was obtained from all individual participants included in the study.

Supplementary material

11060_2017_2547_MOESM1_ESM.doc (55 kb)
Supplementary material 1 (DOC 55 KB)
11060_2017_2547_MOESM2_ESM.jpg (1 mb)
The efficiency of the Sudan B Black (SBB) based autofluorescence quenching protocol is reversible with excess wash and is dependent on the concentration of SBB. As shown in the figure, exposure to SBB at a concentration lower than 0.3% was not sufficient to quench autofluorescence from frozen human brain tissue. In contrast, autofluorescence signals were completely eliminated when the concentration of SBB reached 0.3%. Of note, when the concentration of SBB was between 0.01% and 0.1%, an excess wash with PBS solution lead to the re-appearance of autofluorescence, indicating that SBB quenched tissue autofluorescence by reversibly binding with fluorescent signals. Scale bars: 50 μm (JPG 1030 KB)
11060_2017_2547_MOESM3_ESM.jpg (8.6 mb)
Multiplex immunofluorescence staining revealed that OPC-like tumor cells are the major proliferating population in a human oligodendroglioma sample. (a-e) Representative IF images of a frozen human oligodendroglioma sample co-stained with markers as indicated. Arrows indicate cells stained for multiple markers. (f, g) H&E staining originally suggested a diagnosis of Oligo-Astrocytoma (Grade II) while the FISH assay indicated 1p/19q co-deletion from this sample (not shown), thus yielding the final diagnosis of Oligodendroglioma. Scale bar: 30 μm (JPG 8830 KB)
11060_2017_2547_MOESM4_ESM.jpg (10.3 mb)
Multiplex immunofluorescence staining revealed that OPC-like tumor cells are the major proliferating population in a human diffuse astrocytoma sample. (a-e) Representative IF images of a frozen human diffuse astrocytoma sample co-stained with markers as indicated. (f, g) H&E staining suggested a diagnosis of diffuse astrocytoma (Grade II). Scale bar: 30 μm (JPG 10588 KB)
11060_2017_2547_MOESM5_ESM.jpg (9.4 mb)
Multiplex immunofluorescence staining revealed that OPC-like tumor cells are the major proliferating population in a human anaplastic astrocytoma sample. (a-e) Representative IF images of a frozen human anaplastic astrocytoma sample co-stained with markers as indicated. (f, g) H&E staining suggested the diagnosis of anaplastic astrocytoma (Grade III). Scale bar: 30 μm (JPG 9621 KB)


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Copyright information

© Springer Science+Business Media, LLC 2017

Authors and Affiliations

  1. 1.Department of Pathology and Pathophysiology, School of MedicineZhejiang UniversityHangzhouChina
  2. 2.The Center for Cancer Research, School of MedicineZhejiang UniversityHangzhouChina
  3. 3.Department of Neurosurgery, Second Affiliated Hospital, School of MedicineZhejiang UniversityHangzhouChina
  4. 4.Department of Pathology, Second Affiliated Hospital, School of MedicineZhejiang UniversityHangzhouChina
  5. 5.Department of Radiology, Second Affiliated Hospital, School of MedicineZhejiang UniversityHangzhouChina
  6. 6.Department of Pathology, Women’s Hospital, School of MedicineZhejiang UniversityHangzhouChina
  7. 7.China Brain BankZhejiang UniversityHangzhouChina
  8. 8.Brain Research InstituteZhejiang UniversityHangzhouChina
  9. 9.Collaborative Innovation Center for Brain ScienceZhejiang UniversityHangzhouChina

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