in situハイブリダイゼーション法2色全体のマウントは、関心の1つまたは2つの遺伝子を用いて、遺伝子発現の空間的および時間的パターンの両方を決定するために使用されます。アプリケーションでは、新規遺伝子の遺伝子発現パターンの評価(例えば1,2、だけでなく、遺伝子発現、次の侮辱の変化(胚操作3、ビーズ4、RNAまたはDNAのエレクトロポレーション5構築)が含まれ…
Disclosures
The authors have nothing to disclose.
Acknowledgements
この作品は、RHFへマーガレットM. Alkek財団によってサポートされていました。
Materials
Material Name
Type
Company
Catalogue Number
Comment
Eggs
Animal
Charles River
Premium Fertile
Stereomicroscope
Microscope
Leica Microsystems
MZ9.5
Marsh Automatic Incubator
Tool
Lyon
RX
Hybridization Incubator
Tool
Hybaid
Micro-4
Adams Nutator orbital mixer
Tool
Fisher
14-062
Curved Forceps (1)
Tool
Electron Microscopy Sciences
72991-4C
Fine scissors
Tool
Fine Science Tools
14161-10
Spoon
Tool
Fine Science Tools
10370-18
Pasteur Capillary Pipette
Electron Microscopy Sciences
70950-12
Microcapillary tube
Sigma
P1049-1PAK
Pull using vertical micropipette puller; blunt end with fine forceps
Microdissecting knife
Fine Science Tools
10056-12
Use to puncture cavities prior to in situ hybridization
Minuten pins 0.2mm diam
Fine Science Tools
26002-20
Keep pins together using a magnetic stirrer;
Sylgard 184 Silicon Elastomer Curing Agent and Base
Reagent
Dow Corning
0001986475
Mix 1 part Curing Agent, 9 parts Base; set O/N at 37C
Diethylpyrocarbonate (depc)
Reagent
Acros Organics
10025025
Add 1ml depc to 1l PBS; shake; autoclave
16% PFA
Reagent
Electron Microscopy Sciences
15710
Tween-20
Reagent
Sigma
P1379-100ML
Proteinase K
Sigma
P2308-5MG
Stock at 10 mg/ml in depc-H2O in 10ul aliquots. Store at -20C.
Formamide, deionized
Reagent
Ambion
9342
Yeast tRNA-25 mg
Reagent
Invitrogen
15401-011
Stock at 20 mg/ml in depc-H2O in 125ul aliquots. Store at -20C
Heparin Sodium salt
Reagent
Sigma
H4784-250MG
Stock at 50 mg/ml in depc-H2O in 100ul aliquots. Store at -20C.
SSC x20 pH5.5
Reagent
Fisher
PR-V4261
EDTA (0.5M)
PR-V4231
Fisher
SDS
Reagent
Fisher
BP166-100
Maleic acid
Reagent
Fluka
63189
Boehringer Blocking Reagent
Reagent
Roche Applied Sciences
11096176001
Use at 2% in MABT. To make, heat with MABT for 20 mn at 60C. Can make a stock and store in 1-5 ml aliquots at 20C.
Heat inactivated sheep serum
Reagent
Jackson Immuno
013-000-121
Dissolve in 10 ml H2O; heat 550C, 30 mn. Aliquot at 1ml, -20C.