Neonatal Mouse Ovary Isolation: A Surgical Procedure to Harvest Pair of Ovaries from Neonatal Mouse Model
Published: April 30, 2023
Abstract
Source: et al. Morgan, S. Culture and Co-Culture of Mouse Ovaries and Ovarian Follicles. J. Vis. Exp. (2015).
In this video, we describe a surgical procedure to isolate ovaries from a neonatal mouse.
Protocol
All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. Neonatal Ovary Dissection and Culture
- Place 1 mL of dissection medium (dissolve 3 mg/mL BSA in Leibowitz L15 medium and filter sterilize through a 0.2 µm pore, 25 mm diameter filter) into each sterile glass embryo dish.
- Cull neonatal mouse pups aged between postnatal day 0 and 5, culling by decapitation.
- Grasp the skin covering the abdominal wall using a pair of fine dissection forceps and make a large incision in the skin and body wall. Pull open the incision so the entire abdomen is exposed. The bladder is usually engorged at this stage and can be punctured to make dissection easier.
- Move the guts out of the way using watchmaker forceps. Follow the uterine horns from the bladder up to the kidney on each side. The ovary is located just below the kidney at the top of the uterus and will appear as a cloud-like structure under a dissecting microscope.
- Grasp the ovary gently with watchmaker forceps and use scissors to sever its attachment to the uterus. Transfer the pair of ovaries into embryo dishes containing pre-warmed dissection medium.
- Carry out fine dissection of ovaries under a dissection microscope on a heated stage (37 °C). Use insulin needles to trim away the bursal sac and any excess material including the fallopian tube, until only the ovary remains.
- Transfer each ovary into the well of a culture plate using a finely drawn curved glass pipette.
Açıklamalar
The authors have nothing to disclose.
Materials
| Leibowitz L15 | Invitrogen | 11415049 | |
| αMEM | Invitrogen | 22571020 | Supplied as sodium bicarbonate buffered (2,200 mg/L) |
| Bovine serum albumin | Sigma Aldrich | A9418 | For dissection medium |
| Bovine serum albumin | Sigma Aldrich | A3311 | For culture medium |
| Bovine serum albumin | Sigma Aldrich | A2153 | For coating glass pipettes |
| FSH | Merck Serono | Gonal F | Batch testing is often necessary. Make up stock solution of 1IU/10µL in αMEM and store at -20 °C |
| Syringe filters (25 mm, 0.2 µm) | Greiner | 16532K | Cellulose acetate filter for filtering larger (>5 mL) volumes of medium. |
| Sterile tubes | Greiner | 187261 | |
| 24-well plate | Greiner | 662160 | |
| Insulin needles | BD medical supplies | 037-7606 | 0.33 mm (29 G) + 1 mL |
| Glass embryo dishes | VWR | 720-0579 | Sterilize, then warm before use |
| Glass pipettes | Fisher Scientific | 10209381 | BSA coated before use |
| Acupuncture needles | Acumedic LTD | 30 mm x 0.25 Type C | |
| Formalin solution, neutral buffered, 10% | Sigma Aldrich | HT5014-120mL | |
| Whatman nucleopore membranes | Camlab | WN/110414 | Use shiny surface up, polycarbonate, 13 mm diameter, 8.0 µm pore size |
Etiketler
Bu Makaleden Alıntı Yapın
Neonatal Mouse Ovary Isolation: A Surgical Procedure to Harvest Pair of Ovaries from Neonatal Mouse Model. J. Vis. Exp. (Pending Publication), e20756, doi: (2023).