Apr 16, 2024
Version 3
Tissue H&E Staining | HuBMAP | JHU-TMC V.3
- 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 H&E Staining | HuBMAP | JHU-TMC. protocols.io https://dx.doi.org/10.17504/protocols.io.kqdg3242qv25/v3Version 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: 98293
Keywords: H&E, histology
Funders Acknowledgement:
Institute of Arthritis and Musculoskeletal and Skin Diseases
Grant ID: U54AR081774
National Cancer Institute
Grant ID: U54CA143868
Abstract
The following are guidelines that will have an effective staining window of 2 to 5 minutes. Developed as progressive stains, the intensity of nuclear staining will increase as the time increases. The hematoxylin and eosin stains will have an expected throughput of 2,000-2,500 slides per 500mL bottle. Actual results may vary from lab to lab depending on the staining equipment used, control of carry over into each solution, and length of time stains are left exposed. As a general rule, we recommend changing the hematoxylin and eosin stains once per week if throughput has not been reached. Clarifying and Bluing solutions should be changed more often.
The Leica autostainer is a single fully automated integrated stainer used for standard
H&E staining and operates by progressing each slide through a series of chemical
changes that first deparaffinize and then stains the tissue slides for histologic review. To
use, the machine is powered on and the covers are removed from the staining vessels
every morning and the hematoxylin is filtered daily. The daily number of slides stained is
added to the log sheet beside the stainer, and all the solutions are changed when the
slide tally reaches 250. At the end of the day, the staining vessels are then covered and
the machine is powered off
Materials
The Leica autostainer
Xylene
Ethanol
Hematoxylin
Clarifier
Bluring reagent
Eosin
Before staining
Before staining
Bake unstained slides for 30-60 minutes at 60 degrees Celsius.
Dewaxing
Dewaxing
The following steps require setting up multiple stations with different solutions.
Set 2 stations with Xylene. Submerge the unstained slide for 3 minutes in each station.
Set 2 stations with 100% ethanol. Submerge the slide for a minute in each station.
Set a station with 95% ethanol. Submerge the slide for a minute in each station.
H&E staining
H&E staining
Wash the slide in running warm water for a minute
Wash the slide in running warm water for a minute
Wash the slide in running warm water for a minute
Wash the slide in running warm water for a minute
Set a station with 95% ethanol. Submerge the slide for a minute in each station.
Set 2 stations with 100% ethanol. Submerge the slide for a minute in each station.
Set 3 stations with Xylene. Submerge the dewaxed unstained slide for a minute in each station.
Final Step: Mount and Coverslip with Covermount
Protocol references
Avantik, April 3 2024, Optik Type 1 | Avantik (avantik-us.com)