Disseksjon av Eye-Brain Complex fra Fly Pupae: En metode for å isolere retinal vev

Abstract

Kilde: DeAngelis, M. W., Johnson, R. I. Disseksjon av Drosophila Pupal Retina for Immunohistochemistry, Western Analysis og RNA Isolation. J. Vis. Utt. (2019).

Denne videoen beskriver hvordan du dissekerer og isolerer øye-hjerne-komplekset fra Drosophila-pupper. Den omtalte protokollen demonstrerer prosedyren som gir vev av høy kvalitet, kompatibel med for eksempel immunstaining, samt andre genuttrykkseksperimenter.

Protocol

Denne protokollen er et utdrag fra DeAngelis og Johnson, Disseksjon av Drosophila Pupal Retina for Immunohistochemistry, Western Analysis, og RNA Isolation,   J. Vis. Exp. (2019). 1. Vev forberedelse Sett opp Drosophila-kryss (som beskrevet tidligere i Greenspan, Cold Spring Harbor Laboratory Press, (2004)) eller kulturspesifikke Drosophila-stammer for å få pupper av ønsket genotype. For å sikre at et stort antall pup…

Representative Results

Figur 1: Utvelgelse og kultur av pupper for disseksjon. (A) Vandrende tredje larval instar (L3) larver og pupper finner langs sidene av sunne Drosophila kulturer. (B) Pre-pupae kan identifiseres av deres gjennomsiktige hvite farge som pigment har ennå ikke blitt generert i beskyttende pupal tilfelle. Fremre-bakre og dorsal-ventral akse av puppen er vist i blått. En fuktig bambu…

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