Olfactory Bulb (OB) Transplants
大脑嗅球(OB)移植
发布: 2016年09月05日第6卷第17期 DOI: 10.21769/BioProtoc.1921 浏览次数: 7703
评审: Anonymous reviewer(s)
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参见作者原研究论文
The authors used this protocol in:
Dec 2015
Abstract
Transplantation in mouse brain slices is a powerful tool in order to study axon targeting and migrational events during development. Taking advantage of donors and recipients belonging to different genotypes, this technique allows researchers to assess the contribution of donor and/or recipient tissue by performing various combinations and to study cell-autonomous functions or effects that are influenced by the recipient’s environment (Bastakis, et al., 2015). Here we describe the transplantation procedure on sagittal brain slices containing olfactory bulb (OB). Specifically, we have transplanted the proximal-to-the-cortex part of dorsal OB to the same region on a recipient slice. Transplanted slices can be cultured for up to 3 days before their morphology is disfigured due to growth in 3D. Re-sectioning of these slices allows for a more detailed immunohistochemical analysis.
Materials and Reagents
- Millicell cell culture insert, 30 mm, hydrophilic PTFE, 0.4 µm (Merck Millipore, catalog number: PICM0RG50 )
- Disposable transfer pipettes (SARSTEDT, catalog number: 86.1172.001 )
- 5 ml stripette disposable serological pipette (Sigma-Aldrich, catalog number: CLS4051 )
- 50 ml Falcon tube
- Sterile 24-well plate
- Stainless surgical blade (Swann Morton, Lance, model: No. 24 )
- Vibratome blade (Campden Instruments, catalog number: 752-1-SS )
- 60 mm culture dishes
- Straight triangle insert pin (Fine Science Tools, catalog number: 26007-04 )
- Stainless steel minutien pins 0.10 mm (Fine Science Tools, catalog number: 26002-10 )
- Filters (0.2 μm, 0.45 μm)
- Mouse embryos at embryonic days E13.5-E15.5
- Leibovitz’s L-15 medium (Thermo Fisher Scientific, GibcoTM, catalog number: 11415-064 )
- SeaPlaqueTM low melting temperature agarose (Lonza, catalog number: 50101 )
- Sterile water
- 100 units/ml penicillin-streptomycin (100x) (Thermo Fisher Scientific, GibcoTM, catalog number: 15070-063 )
- NaCl (Merck Millipore, catalog number: 106404 )
- KCl (Merck Millipore, catalog number: 104936 )
- Na2HPO4 (Merck Millipore, catalog number: 106586 )
- NaH2PO4 (Sigma-Aldrich, catalog number: S-0751 )
- NaOH (Merck Millipore, catalog number: 106498 )
- Glucose (Sigma-Aldrich, catalog number: G8270 )
- DMEM/F12 medium (Thermo Fisher Scientific, GibcoTM, catalog number: 21041-025 )
- Fetal bovine serum (FBS) (Merck Millipore, Biochrom, catalog number: S0115 )
- 1x glutamax (Thermo Fisher Scientific, GibcoTM, catalog number: 35050-038 )
- N-2 supplement (100x) (Thermo Fisher Scientific, GibcoTM, catalog number: 17502-048 )
- Neurobasal medium (Thermo Fisher Scientific, GibcoTM, catalog number: 12348-017 )
- B-27 supplement (50x) (Thermo Fisher Scientific, GibcoTM, catalog number: 17504-044 )
- Paraformaldehyde (PFA) (Sigma-Aldrich, catalog number: P6148 )
- Leibovitz’s L-15/penicillin-streptomycin medium (see Recipes)
- 3% SeaPlaque agarose in Leibovitz’s L-15 (see Recipes)
- 10x phosphate buffer solution (PBS) (see Recipes)
- 10 M NaOH (see Recipes)
- Sterile 1x PBS (0.1 M PBS) with 100 units/ml penicillin-streptomycin (see Recipes)
- 25% glucose in 1x PBS (see Recipes)
- DMEM/F12 plus medium (see Recipes)
- Neurobasal/ B-27 plus medium (See Recipes)
- 4% paraformaldehyde (PFA) (see Recipes)
Equipment
- 37 °C, 5% CO2 forced-air incubator (Thermo Fisher Scientific, model: 3110 )
- Dissecting microscope (Leica Microsystems, model: MZ125 )
- 2 Dumont #5 forceps (Fine Science Tools, catalog number: 11251-10 )
- Moria perforated spoon (Fine Science Tools, catalog number: 10370-17 )
- 250 ml glass beaker
- Two-in-One micro spatula (Fine Science Tools, catalog number: 10091-12 )
- Leica vibratome (VT 1000S)
- Nickel plated pin holder (Fine Science Tools, catalog number: 26018-17 )
Note: All surgical tools should be UV-sterilized before use.
Procedure
文章信息
版权信息
© 2016 The Authors; exclusive licensee Bio-protocol LLC.
如何引用
Savvaki, M. and Karagogeos, D. (2016). Olfactory Bulb (OB) Transplants. Bio-protocol 6(17): e1921. DOI: 10.21769/BioProtoc.1921.
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分类
发育生物学 > 细胞生长和命运决定 > 神经元
细胞生物学 > 组织分析 > 组织分离
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