드로소필라 늦은 푸파 간접 비행 근육 (IFM) 해부 : 높은 처리량 조직 수집을위한 방법

Published: April 30, 2023

Abstract

출처: 카오, S. Y., 외. 오믹스 접근에 대한 드로소필라 멜라노가스터 비행 근육의 해부. J. 비스 익스펙. (2019).

Drosophila 비행 근육 근육 개발 및 생리학에 대 한 모델 시스템으로 사용 됩니다. 이 비디오는 성인의 비행 근육을 설명하고 pupae에서 RNA 염기서열에 적합한 간접 비행 근육을 개발하는 해부 프로토콜을 강조합니다.

Protocol

이 프로토콜은 카오 외에서발췌., Omics 접근에 대한 드로소필라 멜라 노가스터 비행 근육의 해부, J. Vis. Exp. (2019). 1. IFM 해부 후 48 h APF 두 #5 생물학 학년 집게, 미세 가위, 표준 유리 현미경 슬라이드, 이중 스틱 테이프, 파이펫, 파이펫 팁, 드라이 아이스 및 (RNA 응용 프로그램의 경우) 격리 시약 (재료의 표참조)을 포함하여 필요한 장비를…

Representative Results

그림 1: 48h APF 후 IFM의 해부. (A)이중 스틱 테이프에 강아지의 정렬. (B)푸팔 케이스로부터 푸파를 제거하고, 케이스를 등등(B’)으로 자르고,강아지(B’)를들어올리는 것으로. 도 2와동일하게 표시된 원 기호입니다. (C)버퍼에 강아지의 전송. (D)</…

Materials

60 mm culture dishes Sigma-Aldrich Z643084-600EA Greiner dishes, 60 mm x 15 mM, vented
Cell phone camera, Samsung Galaxy S9 Samsung SM-G960F/DS used for photos not taken under a microscope
Double stick tape Scotch/3M 3M ID 70005108587 Double-sided tape, available at most office supply handlers
Dumont #5 Forceps Fine Science Tools 11252-20 Inox straight tip 11 cm forceps, Biology grade with 0.05 mm x 0.02 mm tip
fluorescent dissecting microscope camera, Leica DFC310 FX camera Leica www.leica-microsystems.com
Fluorescent dissecting microscope software, Leica Application Suite (LAS) version 4.0.0 Leica www.leica-microsystems.com
Fluorescent dissecting microscope, Leica M165 FC Leica www.leica-microsystems.com
Fly: Bru1[M2] Fly stock; This paper
Fly: Bru1[M3] Fly stock; This paper
Fly: Mef2-GAL4 Bloomington Stock Center BDSC:27390 Fly stock
Fly: salm[1] Bloomington Stock Center 3274 Fly stock
Fly: salm[FRT] Fly stock; see Spletter et al., Elife, 2018
Fly: UAS-Bru1IR Vienna Drosophila Research Center GD41568 Fly stock, RNAi hairpin
Fly: UAS-GFP::Gma Bloomington Stock Center BDSC:31776 Fly stock
Fly: UAS-mCD8a::GFP Bloomington Stock Center BDSC:5130 Fly stock
Fly: w[1118] Bloomington Stock Center 3605 Fly stock
Fly: weeP26 Fly stock; see Clyne et al., Genetics, 2003
GFP detection reagent, GFP-Booster ChromoTek gba488-100
Glycogen Invitrogen 10814-010
Image processing software, Photoshop CS6 Adobe www.adobe.com
Isopropanol Sigma-Aldrich I9516-25ML 2-propanol
Method 1 (RNA isolation): TRIzol Life Technologies 15596018 Guanidinium isothiocyanate and phenol monophasic solution
Method 2 (RNA isolation): Method 1 + TURBO DNA-free Kit Invitrogen AM1907 TRIzol isolation followed by treatment with a kit to remove DNA
Method 3 (RNA isolation): Direct-zol RNA Miniprep Plus Kit Zymo Research R2070S RNA isolation in TRIzol, but over commercial columns instead of using phase separation.
Recommended DNase treatment performed with Monarch Dnase I in Monarch DNase I Reaction buffer.
Method 4 (RNA isolation): RNeasy Plus Mini Kit Qiagen 74134 We used the provided DNase treatment. IFM pellets were homogenized in RTL buffer as suggested for animal tissues.
Method 5 (RNA isolation): ReliaPrep RNA Tissue Miniprep System Promega Z6110 We applied the protocol for 'Purification of RNA from Fibrous Tissues'.
Method 6 (RNA isolation): Monarch Total RNA Miniprep Kit New England Biolabs T2010G We applied the protocol for tissues/leukocytes and lysed in 300 µL of RNA Protection Reagent.
Microcentrifuge tubes Thermo Fisher AM12400 RNase-free Microfuge Tubes, 1.5 mL
Microscope slides Thermo Fisher 12342108 Standard slides, uncharged, 1 mm
Paintbrush Marabu 1910000000 Marabu Fino Round No. 0, or similar brush from any art supply
Paraformaldehyde Sigma-Aldrich 158127
PBS buffer (1x) Sigma-Aldrich P4417 Phosphate buffered saline tablets for 1 L solutions, pH 7.4
PFA PureTip Pipette Tips Elemental Scientific ES-7000-0101 Optional substitute for standard pipette tips to reduce sample loss; 100 mL, 0.8 mm orifice
Pipette tips Sigma-Aldrich P5161 Universal 200 mL pipette tips
RNA concentration Approach 1 & RNA integrity traces, Bioanalyzer Agilent Technologies G2939BA
RNA concentration Approach 2, Nanodrop Thermo Fisher ND-2000
RNA concentration Approach 3, Qubit 4 Fluorometer Invitrogen Q33238
RNase A Promega A7937
RNase-free water, Diethyl pyrocarbonate (DEPC) Sigma-Aldrich D5758 DEPC treat water overnight and then autoclave, to remove all RNase.
RT Kit #1: Super Script III Reverse Transcriptase Kit Invitrogen 18080-044 reverse transcription kit
RT Kit #2: LunaScript New England Biolabs E3010S reverse transcription kit
RT Kit #3: QuantiNova Reverse Transcription Kit Qiagen 205410 reverse transcription kit
Statistical software: GraphPad Prism GraphPad Prism www.graphpad.com
Statistical software: Microscoft Excel Microsoft Purchased as part of the bundle: Office Home & Student 2019
Table-top centrifuge Eppendorf 5405000760 Eppendorf Centrifuge 5425 or equivalent
Tissue/ Kimwipes Sigma-Aldrich Z188956 Standard tissue wipes
Transfer pipette Sigma-Aldrich Z350796 Plastic pipette
Vannas spring scissors Fine Science Tools 15000-00 3 mm cutting edge, tip diameter 0.05 mm, length 8 cm
Whatman paper Sigma-Aldrich 1004-070 Filter paper circles, Grade 4, 70 mm

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Cite This Article
Drosophila Late Pupa Indirect Flight Muscle (IFM) Dissection: A Method for High-Throughput Tissue Collection. J. Vis. Exp. (Pending Publication), e20128, doi: (2023).

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