Apr 16, 2024
Version 2
Tissue Fixation | HubMAP | JHU-TMC V.2
- Kyu Sang Han1,
- Pei-Hsun Wu1,
- Joel Sunshine2,
- Ashley Kiemen2,
- Miklhail James2,
- Sashank Reddy2,
- Denis Wirtz1,2
- 1Johns Hopkins University;
- 2Johns Hopkins Medicine
Protocol Citation: Kyu Sang Han, Pei-Hsun Wu, Joel Sunshine, Ashley Kiemen, Miklhail James, Sashank Reddy, Denis Wirtz 2024. Tissue Fixation | HubMAP | JHU-TMC. protocols.io https://dx.doi.org/10.17504/protocols.io.3byl4929jgo5/v2Version created by Kyu Sang Han
Manuscript citation:
A.M. Braxton, A.L. Kiemen, M.P. Grahn, A. Forjaz, J. Parksong, J.M. Babu, J. Lai, L. Zheng, N. Niknafs, L. Jiang, H. Cheng, Q. Song, R. Reichel, S. Graham, A.I. Damanakis, C.G. Fischer, S. Mou, C. Metz, J. Granger, X.-D. Liu, N. Bachmann, Y. Zhu, Y.Z. Liu, C. Almagro-Pérez, A.C. Jiang, J. Yoo, B. Kim, S. Du, E. Foster, J.Y. Hsu, P.A. Rivera, L.C. Chu, D. Liu, E.K. Fishman, A. Yuille, N.J. Roberts, E.D. Thompson, R.B. Scharpf, T.C. Cornish, Y. Jiao, R. Karchin, R.H. Hruban, P.-H. Wu, D. Wirtz, and L.D. Wood, “3D genomic mapping reveals multifocality of human pancreatic precancers”, Nature (2024)
A.L. Kiemen, A. Forjaz, R. Sousa, K. Sang Han, R.H. Hruban, L.D. Wood, P.H. Wu, and D. Wirtz, “High-resolution 3D printing of pancreatic ductal microanatomy enabled by serial histology”, Advanced Materials Technologies 9, 2301837 (2024)
T. Yoshizawa, J. W. Lee, S.-M. Hong, D.J. Jung, M. Noe, W. Sbijewski, A. Kiemen, P.H, Wu, D. Wirtz, R.H. Hruban, L.D. Wood, and K. Oshima. “Three-dimensional analysis of ductular reactions and their correlation with liver regeneration and fibrosis”, Virchows Archiv (2023).
A.L. Kiemen, A.I. Damanakis, A.M. Braxton, J. He, D. Laheru, E.K. Fishman, P. Chames, C. Almagro Perez, P.-H. Wu, D. Wirtz, L.D. Wood, and R. Hruban, “Tissue clearing and 3D reconstruction of digitized, serially sectioned slides provide novel insights into pancreatic cancer”, Med 4, 75-91 (2023)
A. Kiemen, Y. Choi, A. Braxton, C. Almagro Perez, S. Graham, M. Grahm, N., N. Roberts, L. Wood, P. Wu, R. Hruban, and D. Wirtz, “Intraparenchymal metastases as a cause for local recurrence of pancreatic cancer”, Histopathology 82: 504-506 (2022)
A.L. Kiemen, A.M. Braxton, M.P. Grahn, K.S. Han, J.M. Babu, R. Reichel, A.C. Jiang, B. Kim, J. Hsu, F. Amoa, S. Reddy, S.-M. Hong, T.C. Cornish, E.D. Thompson, P. Huang, L.D. Wood, R.H. Hruban, D. Wirtz and P.H. Wu, “CODA: quantitative 3D reconstruction of large tissues at cellular resolution”, Nature Methods 19: 1490-1499 (2022)
K.S.Han, I. Sander, J. Kumer, E. Resnick, C. Booth, B. Starich, J. Walston, A.L. Kiemen, S. Reddy, C. Joshu, J. Sunshine, D. Wirtz, P.-H. Wu "The microanatomy of human skin in aging." bioRxiv (2024): 2024-04.
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: April 16, 2024
Last Modified: April 16, 2024
Protocol Integer ID: 98294
Funders Acknowledgement:
Institute of Arthritis and Musculoskeletal and Skin Diseases
Grant ID: U54AR081774
National Cancer Institute
Grant ID: U54CA143868
Disclaimer
The protocols.io team notes that research involving animals and humans must be conducted according to internationally-accepted standards and should always have prior approval from an Institutional Ethics Committee or Board.
Abstract
First and most important - the original tissue sample must be of good quality. Factors such as warm
ischemic time, the time delay between tissue excision and fixation, etc. are important. Ideally, tissues
should be acquired as close to still being viable as possible, and put into fixative as soon as possible
following excision. Delays lead to cell death, autolysis, and loss of tissue and cell integrity with concomitant
losses of immunostaining (e.g., due to proteolysis of the antigen). If acquiring animal tissues, consider
performing perfusion fixation before organ/tissue removal if it is an option
Tissue Fixation
Tissue Fixation
We submerge the biopsy in the histology container prefilled with 10% NBF for fixation
Minimizing the warm ischemic time is critical. The time delay between tissue excision and fixation is detrimental to further analysis.
Note on the effect of delay to formalin fixation - Delay to
formalin fixation effect on breast biomarkers - PubMed (nih.gov)
The rate of penetration of formaldehyde depends on the size of the biopsy. Trimming is often required to facilitate fixation. Tissues placed in the tissue cassettes should be no thicker than 3-4mm because the interior of a specimen may not become fully fixed, or significant autolysis can occur.
Tissue Trimming
Tissue Trimming
Trimming also helps remove unwanted parts of the biopsy such as excessive fat and traumatized
edges.
Label tissue cassette with sample ID with pencil
Place the trimmed tissue in cassette
Place cassettes back into the histology container
Tissue fixation (continue)
Tissue fixation (continue)
Refill 10% NBF into the container and leave for 12~48 hours at room temperature.
Use plenty of fixative. The general rule is to use at least 15 volume equivalents of formalin per volume
of tissue. A higher formalin-to-tissue ratio certainly won't hurt, and just requires a larger container.
Formalin is relatively cheap, so don’t skimp on this step.
Gentle agitation of the tissue in the formalin during fixation maximize diffusion and reduce the poor local fixation.
Pour the formalin into an “excess formalin” waste container in the hood.
Rince the cassettes in the histology container with 1x PBS
Drain and refill the PBS. Leave the cassette submerged in PBS and store at 4 degree C
until processing.
Properly fixed tissue can be stored in PBS up to a week.
Protocol references