Jul 27, 2023
Human ovarian tissue fixation
- hannah.anvari1,
- Pooja Devrukhkar1,
- francesca.e.duncan duncan1
- 1Northwestern Medicine OBGYN, Northwestern University
Protocol Citation: hannah.anvari, Pooja Devrukhkar, francesca.e.duncan duncan 2023. Human ovarian tissue fixation. protocols.io https://dx.doi.org/10.17504/protocols.io.x54v9pyz1g3e/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: July 27, 2023
Last Modified: July 27, 2023
Protocol Integer ID: 85594
Keywords: ovary, fixation, FFPE, explant
Funders Acknowledgement:
SenNet
Grant ID: U54AG075932
SenNet
Grant ID: 1UG3CA268105
Abstract
Purpose: This protocol is intended for use in fixing human ovarian tissue in a research setting. This protocol supplements the steps detailed in the collection, processing, and long-term storage of human ovarian tissue as formalin-fixed paraffin-embedded (FFPE) samples in protocol: dx.doi.org/10.17504/protocols.io.e6nvwdk29lmk/v1.
Guidelines
Researchers working with human specimens will adhere to all safety and training protocols required by the institution (Northwestern Medicine/Northwestern University) including but not limited to:
- Biosafety Certification
- Bloodborne Pathogens Certification
- Working with Formaldehyde Certification
- Collaborative Institutional Training Initiative (CITI program) certification
Materials
- Modified Davidson’s Fixative, MDF (Electron Microscopy Sciences, 64133-50)
- Histology grade ethanol (Fisher Scientific 04-355-223)
- Invitrogen™ RNase-free Microfuge Tubes (ThermoFisher Scientific, AM12400)
Safety warnings
Researchers will wear personal protective equipment when working with human specimens, including gloves, masks, and lab coats.
Ethics statement
This protocol takes place under approved IRB protocol through Northwestern University (NU12G09) for collection of human ovarian tissue through Northwestern Medicine.
Ovarian tissue fixation for fixed wedges and ovarian explant pieces
Ovarian tissue fixation for fixed wedges and ovarian explant pieces
Fill 1.5 mL micro centrifuge tube with 1 mL of Modified Davidson’s Solution (Cat. #64133- 50; Electron Microscopy Sciences, Hatfield, PA)
Add one ovary wedge or all tissue pieces (ovarian explant) from one experimental well per 1.5 mL micro centrifuge tube.
Allow all tubes to rock @ room temperature (RT) for 2-4 hrs.
Transfer tubes to cold room and rock at 4°C overnight (Fig. 1).
Fig. 1. Ovarian tissue wedges resting on rocker at 4°C overnight (A). Ovarian tissue wedges are embedded in paraffin blocks so that the cortex and medulla will be present in every section (B). A representative tissue section is shown for the block in B which includes both cortex and medulla approximated by a black line (C). Ovarian explants from one tissue well can be embedded together so that they are sectioned in one plane (D).
Wash in 1 mL 70% EtOH 3x10 minutes, rocking in between each, and then store in 70% EtOH @ 4°C until processing/paraffin embedding, no longer than 1 week.
Ovarian tissue wedges containing both cortex and medulla should be embedded so that the cortex and medulla will be sectioned in one plane (Fig. 1B). A representative H&E tissue section indicates the proper orientation (Fig. 1C).
Ovarian tissue pieces from explant culture should be embedded together, if cultured in the same well (Fig. 1D). While embedding try to ensure that tissues sit as evenly as possible, so that they are sectioned in the same plane.