Oct 11, 2022
Tissue Fixation Preparation
- Stephen Fisher1,
- Marielena Grijalva1,
- Rong Guo1,
- sarahjoh 1,
- Hieu Nguyen1,
- John Renz2,
- Jean G Rosario1,
- Steven Rudich2,
- Brian Gregory1,
- Junhyong Kim1,
- Kate O'Neill1
- 1University of Pennsylvania;
- 2Gift of Life Donor Program
Protocol Citation: Stephen Fisher, Marielena Grijalva, Rong Guo, sarahjoh , Hieu Nguyen, John Renz, Jean G Rosario, Steven Rudich, Brian Gregory, Junhyong Kim, Kate O'Neill 2022. Tissue Fixation Preparation. protocols.io https://dx.doi.org/10.17504/protocols.io.6qpvr61ozvmk/v1
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: August 13, 2022
Last Modified: October 11, 2022
Protocol Integer ID: 68610
Keywords: tissue fixation preparation this protocol, tissue fixation preparation, fixation of tissue, tissue fixation, fixed tissue, preparation for pathology review, degradation of the tissue, chemical fixative, used chemical fixative, tissue quality, formaldehyde, paraformaldehyde, fixation, pathology review, tissue, follicular morphology, preferred fixative, tissue of interest, other fixative, various reagent, pathology, preparation, pathologist review, reagent
Funders Acknowledgements:
NIH
Grant ID: U54HD104392
Abstract
This protocol describes fixation of tissue in preparation for pathology review to assess tissue quality.
Prior to performing molecular analyses, it is critical to have experienced pathologist confirm the tissue of interest is captured, the region of interest is represented and there are no abnormalities. Fixed tissue is preferred for pathologist review as it prevents autolysis and degradation of the tissue, cells and cellular components so they can be observed both anatomically and microscopically in their most natural state following sectioning. Various reagents can be used for fixation. Formaldehyde is a water-based solution and is the most commonly used chemical fixative. Paraformaldehyde has been found to be a preferred fixative for the ovary as it maintains oocyte and follicular morphology which is often disrupted with other fixatives.
Materials
- Freshly dissected tissue block
- Ice
- 70% EtOH (made fresh; kept at 4°C)
- Ice bucket
- 1.5 mL LoBind tubes EppendorfCatalog #022431021
- 4% sucrose in 1X DPBS; kept at 4°C
DPBS (10X), no calcium, no magnesium Thermo Fisher ScientificCatalog #14200075
- 15mL conical tubes with 10% buffered formalin
- 15mL conical tubes with 4% paraformaldehyde (PFA) (fresh for 1 week, keep on ice)
Eppendorf Conical Tube 15mLMerck MilliporeSigma (Sigma-Aldrich)Catalog #EP0030122151
Troubleshooting
4% PFA Tissue Fixation of Ovaries
4d
Place each pre-weighed piece of tissue in either a 1.5mL Eppendorf tube or 1.5mL cryo tube.
Keep PFA tubes on ice. Transfer tissue specimen into a tube and refrigerate at 4 °C for 24:00:00 .
1d
After 24:00:00 , transfer tissue into 4% sucrose/PBS solution and incubate for 24:00:00 at 4 °C .
2d
After 24:00:00 , transfer tissue specimen to 70% EtOH (freshly made) and store at 4 °C until ready for paraffin embedding.
1d
Proceed with paraffin embedding of samples within 3 weeks.
Paraffin embedded tissue can now be used for H&E Pathology Review.
Formalin Tissue Fixation of Fallopian Tube, Uterine Body, and Cervix
2d
Place each pre-weighed piece of tissue in either a 1.5mL Eppendorf tube or 1.5mL cryo tube.
Place tissue specimen in a prepped and labeled tube. Keep at room temperature for 24:00:00 .
1d
After 24:00:00 , transfer tissue specimen to 70% EtOH (freshly made) and store at 4 °C until ready for paraffin embedding.
1d
Proceed with paraffin embedding of samples within 3 weeks.
Paraffin embedded tissue can now be used for H&E Pathology Review.