Apr 25, 2022

Public workspaceHuman Pregnant Fallopian Tube Tissue Collection and Preservation Methods - UCSD Female Reproductive TMC

DOI
dx.doi.org/10.17504/protocols.io.dm6gpbd5plzp/v1
  • 1University of California, San Diego
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DOI: dx.doi.org/10.17504/protocols.io.dm6gpbd5plzp/v1
Protocol Citation: Valentina Stanley, Scott Lindsay-Hewett, Mana Parast, Louise Laurent 2022. Human Pregnant Fallopian Tube Tissue Collection and Preservation Methods - UCSD Female Reproductive TMC. protocols.io https://dx.doi.org/10.17504/protocols.io.dm6gpbd5plzp/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: April 14, 2022
Last Modified: April 25, 2022
Protocol Integer ID: 60813
Keywords: HuBMAP, UCSD Female Reproductive TMC
Abstract
Human pregnant fallopian tube tissue collection and storage protocol for HuBMAP's UCSD Female Reproductive TMC.
Materials
  1. Sterile surgical dissecting instruments (scissors/forceps)
  2. Gauze pad
  3. PBS (cold)
  4. Liquid Nitrogen
  5. Empty microfuge tubes x8
  6. Microfuge tubes pre-filled with RNAlater x8
  7. 10% Formalin filled tubes x4
Preparation
Preparation
Fallopian tubes should be collected into empty sterile cups right after sterilization (salpingectomy or tubal ligation). Keep left and right tubes separate.
Take a photo of the tubes prior to sampling. If pieces of the tissue were collected for clinical pathological examination, maintain the original orientation of tubes from the fimbrial (distal) end to the tubal (proximal) end after such sampling.


Wash the tissues in cold PBS while maintaining the original orientation.
Preserving the fimbria
Preserving the fimbria
Separate the fimbriated end from the tube (if not already separated).
Divide the fimbriated end lengthwise into 2 or 3 equal sized sections (depending on size).
Place one fimbriated piece into a 10% formalin-filled tube (1st priority), the other into RNAlater (2nd priority), and the third to be snap-frozen (3rd priority).
Preserving the tube
Preserving the tube
Collect cross sections of the tube into 10% formalin (x1), RNAlater (x3) and to be snap-frozen (x3). Each piece should be about 5mm in length, depending on the total length of the tube. If there is an excess of tissue, the formalin piece(s) can be larger.
Collect the formalin-preserved piece from the most distal end (closest to the fimbria).
For the right tube, collect the RNAlater pieces from the center of the tube and the snap frozen pieces from the proximal end. T1 should be most distal, then T2, then T3 should be most proximal.


For the left tube, collect the snap frozen pieces from the center of the tube and the RNAlater pieces from the proximal end. T1 should be most distal, then T2, then T3 should be most proximal.


Flash-freezing
Flash-freezing
Drop the snap frozen labeled tubes into liquid Nitrogen. Leave for ~2-10 minutes, then store in -80C freezer.
Processing with RNAlater
Processing with RNAlater
Place RNAlater tubes into a 4C fridge. Let these sit for 24-48 hours, then remove the RNAlater with a sterile transfer pipette and store in a -80C freezer.
Formalin fixing
Formalin fixing
After one week in formalin, place each formalin-fixed piece into a labeled cassette to be processed into FFPE blocks.