플라이 푸파에서 눈 -뇌 복합체의 해부 : 망막 조직을 격리하는 방법

Abstract

출처: DeAngelis, M. W., 존슨, R. I. 면역 작용, 서양 분석 및 RNA 격리를 위한 Drosophila Pupal 망막의 해부. J. 비스 익스펙. (2019).

이 비디오는 Drosophila pupae에서 눈-뇌 복합체를 해부하고 격리하는 방법을 설명합니다. 특징적인 프로토콜은 고품질 조직을 산출하는 절차를 시연하며, 예를 들어 면역 염색뿐만 아니라 다른 유전자 발현 실험과 호환됩니다.

Protocol

이 프로토콜은 면역 집중화학, 서양 분석 및 RNA 격리를 위한 Drosophila Pupal 망막의 DeAngelis와 Johnson, 해부,   J. Vis. Exp. (2019)에서 발췌한 것입니다. 1. 조직 준비 드로소필라 십자가(이전에 그린스펀,콜드 스프링 하버 실험실 프레스, (2004)에 설명된 대로 설정하거나, 원하는 유전자형의 새끼를 얻기 위해 특정 드로소필라 균주?…

Representative Results

그림 1: 해부에 대한 강아지의 선택과 문화. (A)건강한 드로소필라 문화의 측면을 따라 세 번째 애벌레 인스타(L3)를 방황하는 애벌레와 푸파를 찾아보고 있습니다. (B)안료가 보호 pupal 케이스에서 생성되지 않은 바와 같이 프리 푸파는 반투명 백색색으로 식별 될 수있다. 푸파의 전방 후방 ?…

Materials

Bamboo splints, 6"	Ted Pella Inc	116
Black dissecting dish			Glass petri dish filled to rim with SYLG170 or SYLG184 (colored black with finely ground charcoal powder). Leave at room temperature for 24-48 h to polymerize.
Blade holder	Fine Science Tools	10053
Bovine serum albumin	Sigma-Aldrich	A7906
Double-sided tape	3M	665
Drosophila& food media, nutrient-rich			7.5% sucrose, 15% glucose, 2.5% agar, 20% brewers yeast, 5% peptone, 0.125% MgSO4.7H2O, 0.125% CaCl2.2H20
Drosophila& food media, standard			Bloomington Drosophila Stock center cornmeal recipe. (https://bdsc.indiana.edu/information/recipes/bloomfood.html)
Fixative solution			4% formadehyde in PBS, pH 7.4.
Forceps	Fine Science Tools	91150-20	Forceps should be sharpened frequently.
Formaldehyde	Thermo Scientific	28908
Glass 9-well dishes	Corning	7220-85	Also known as 9-well dishes
Glass coverslips (22 x 22 mm)	Fisher Scientific	12-542-B
Glass microscope slides (25 x 75 x 1 mm)	Fisher Scientific	12-550-413
Glass petri dish	Corning	3160-100BO
Glycerol	Sigma-Aldrich	G5516
Microcentrigure tubes	Axygen	MCT-175-C
Microdissection scissors	Fine Science Tools	15000-03
Microwell trays (72 x 10 µL wells)	Nunc	438733
Mounting media			0.5% N-propylgallate and 80% glycerol in PBS
N-propylgallate	Sigma-Aldrich	P3130
PBS (phosphate buffered saline pH 7.4)	Sigma-Aldrich	P5368	Prepare according to manufacturer's instructions
PBT			0.15% TritonX and 0.5% bovine serum albumin in PBS, pH 7.4
Pin holder	Fine Science Tools	26016-12
Primary antibody: goat anti-GAPDH	Imgenex	IMG-3073	For Western blotting. Used at 1:3000
Primary antibody: rabbit anti-cleaved Dcp-1	Cell signaling	9578S	For immunofluorescence. Used at 1:100
Primary antibody: rat anti-DEcad	Developmental Studies Hybridoma Bank	DCAD2	For immunofluorescence. Used at 1:20
Primary antibody: rat anti-DEcad	DOI: 10.1006/dbio.1994.1287	DCAD1	Gift from Tadashi Uemura. Used at 1:100.
Scalpel blades	Fine Science Tools	10050	Break off small piece of scapel blade and secure in blade holder.
Secondary antibody: 488-conjugated donkey anti-rat IgG (H+L)	Jackson ImmunoResearch	712-545-153	For immunofluorescence. Used at 1:200
Secondary antibody: cy3-conjugated goat anti-rabbit IgG (H+L)	Jackson ImmunoResearch	111-165-144	For immunofluorescence. Used at 1:100
Secondary antibody: HRP-conjugated goat anti-rat IgG (H+L)	Cell Signaling Technology	7077	For Western blotting. Used at 1:3000
Secondary antibody: HRP-conjugated rabbit anti-goat IgG (H+L)	Jackson ImmunoResearch	305-035-003	For Western blotting. Used at 1:3000
Stereo dissecting microscope (M60 or M80)	Leica Microsystems		or similar microscope
Sylgard (black)	Dow Corning	SYLG170
Sylgard (transparent)	Dow Corning	SYLG184	Color black with finely ground charcol powder
Tissue: Kimwipes	KIMTECH	34120
TritonX	Sigma-Aldrich	T8787
Tungsten needle, fine	Fine Science Tools	10130-10	Insert into pin holder
Tungsten needle, sturdy	Fine Science Tools	10130-20	Insert into pin holder
Yeast paste	(local supermarket)		Approximately 2 tablespoons Fleischmann's ActiveDry Yeast (or similar) dissolved in ~20 mL distilled H2O