Antibodies that Work In Tribolium Castaneum

Kevin Burns5/20/2011

Notes on this Document• For antibodies I’ve used personally, the titles contain the following information

– Original antigen used to produce antibody– A rough guess at the stage/stages of Tc shown– Antibody procurement info, e.g. DSHB, Supplier, or Lab– Host species and working dilution

• I have often thrown in co-stains to further illustrate morphology and to make things look cool. However, mostly this is just confusing.– KT817 is an enhancer trap line from Sue Brown that marks oenocytes. It is also cool for marking

A1-A8

• The second half of this document in-exhaustively covers antibodies that have been used in published works, normally on beetle, but occasionally on other arthropods, please update me if you find any new ones (Kevin.Burns@cchmc.org)

• There is a PDF and a Powerpoint version of this document (The hyperlinks work in the PPT), if your email server does not support attachments of such & such size, I will post this document on http://burnska08.wordpress.com/kevin-burns-home-page/

Index• AbdA (+UBX)• AntP• Futsch (22c10)• HNF4• Notch Intracellular Domain• Orthodenticle (GP / Rat)• Phospho-Histone3• Phospho-MAPK• Spalt• Trachea (a2A12)• UBX (see AbdA)

Index of Other’s Results

Dm Abd-A/UBXPre-Hatching, ~64h at 30C

DSHB: Mouse Monoclonal, 1:5

UBX/AbdA UBX/AbdADAPI KT817-YFP

Index for KB’s Antibodies

Dm AntPGermband retraction 30h @30C

DSHB: Mouse Monoclonal, 1:25+

NICD

AntP

DAPI

KT817-YFP

AntP

Please advise naïve young investigators that AntP is not just expressed in thoracic segments but also in the CNS Index for KB’s Antibodies

Dm En/InvDorsal closure/Leg articulation (36h)

DSHB: 4D9Mouse Monoclonal, 1:25

En/InvSpalt En/Inv

Index for KB’s Antibodies

Tc Engrailed

Cocalico/Gebelein, coming in JulyRat/Guinea Pig

Kevin Burns is making this in Brian Gebelein’s lab in order to do some co-stains with the multitude of mouse antibodies

Index for KB’s Antibodies

Dm FutschDorsal closure/48h? (Ventral View)

DSHB: 22c10Mouse Monoclonal, 1:50

22c10

Index for KB’s Antibodies

HNF448h @ 30C

Cocalico/Gebelein: Rat Serum, 1:1000

HNF4 HNF4KT817-YFP

p

Mesodermal staining (arrowheads), continues underneath more superficial oenocyte staining (e.g. red circle)p = pleuropod in A1 Index for KB’s Antibodies

Notch Intracellular DomainGermband extension

DSHB: C17.9C6Mouse Monoclonal 1:5

NICD NICDDAPI

Index for KB’s Antibodies

Dm OTD full lengthMix sample

Cocalico/Cook: Rat Serum, 1:500

OTD

I am conducting another stain to see if this detects the OTD that replaces bicoid in early patterning…

Index for KB’s Antibodies

Dm OTD full lengthMix sample

Cocalico/Cook: Guinea Pig Serum, 1:500

OTD

Index for KB’s Antibodies

phospho-Histone 3Full extension

Millipore: 05-806Mouse, 1:1000

pH3 pH3 DAPI

Index for KB’s Antibodies

Di-Phosphorylated MAPKpost-gastrulation and full germband

Sigma: M-8159Mouse Monoclonal, 1:50

pERKpERK

Index for KB’s Antibodies

Dm Spalt Major24h, 36h @ 30C

Cocalico/Gebelein: Rabbit Serum, 1:1500

SpaltSpalt

pERKSpalt

KT-817-YFP

Spalt

Index for KB’s Antibodies

Dm unknown (tracheal System)Pre-HatchingDSHB: a2A12

Mouse IGM Monoclonal, 1:25

NICD

a2A12DAPI

This stain was achieved with IgG secondary antibodies, my guess is secondaries designed against IgM would fare much better

Index for KB’s Antibodies

Table of Kevin’s Antibodies

Cat#/name Antigen Host Isotype Concentration Stains what Available From

4C3 Dm AntP M IgG 1:50Thoracic segments, ventral nervous system DSHB

22c10 Dm Futsch M IgG 1:50 CNS commissures only (so far) DSHB

HNF4 Tc HNF4 Rat Serum 1:2000Malpighian tubules, hindgut, fat bodies, oenocytes Brian Gebelein

FP6.87 Dm AbdA/Ubx M IgG 1:5

T3/Abdominal segments less those in AbdB domain, Recognizes both AbdA and Ubx DSHB

A2a12 Dm? M IgM 1:25 Tracheal system DSHBSpalt 26 Dm Spalt Rabbit Serum 1:1500 Ectodermal streak Brian Gebelein

4D9 Dm Engrailed M IgG 1:10+ Posterior segmental boundaries DSHB2B8 Dm Eve M DSHB

C17.9C6 Dm NICD M IgG11:5 better than 1:25 Notch Receiving Cells DSHB

M-8159 pErk M 1:50 Di-phosphorylated MAPK Sigma pH3 M 1:1000 easily Mitotic cells Millipore Caspase3 M Apoptotic cells Cell SignalingG6 Dm Otd Guinea Pig serum 1:500- Ventral CNS Tiffany CookR2 Dm Otd Rat serum 1:500- Ventral CNS Tiffany Cook

Index for KB’s Antibodies

Index of Other People’s • Caudal • Caspase 3• Cx (scr)• DC0 (mushroom body etc) • DLL • engrailed 4F11 from DSHB• Even-skipped• Nubbin (Drosophila + Crustacean + Chelicerates) • Prd, Gooseberry, Gooseberry-Neuro (Pairberry)• pSMAD • pY• Synapsin 1

Index for KB’s Antibodies

CaudalShulz&Tautz 1998 Dev Genes & Evolution

Generation of an antibodyPart of the cadA ORF (open reading frame, corresponding to aminoacids 79–249) was amplified by PCR and cloned in frame intothe EcoRV site of the pRSET-C (Invitrogen) vector. The

Index of Other’s Results

Caspase 3Aranda&Tautz2009 Dev Genes Evol

For apoptosis detection using the anti cleaved caspase3 antibody (Cell Signalling Technology, Inc.)

Index of Other’s Results

Cx (Scr)Shippy & Denell 2006 Genetics

A cross-reacting polyclonal antibody to Drosophila Scr, α-DmScr (a gift from Thomas Kaufman), was used to detect Cx

Index of Other’s Results

DC0Zhao&Farris 2008

Anti-DC0 primary antibody, a polyclonal antibodyagainst the catalytic subunit of Drosophila melanogasterprotein kinase A, was a gift provided by Dr. Daniel Kalderon.This antibody has been shown to have a high affinityfor mushroom body intrinsic neurons in a range of insectspecies (Skoulakis et al., 1993; Farris, 2005a,b). Index of Other’s Results

DLLe.g. Panganiban&Carroll 1997 PNAs

• Butterfly Staining…

Production and characterization of the Dll homeodomain antibody was described in Panganiban et al. (4)

Index of Other’s Results

Engrailed 4F11 (alternate DSHB Clone)e.g. Pueyo&Couso2008 PNAS

This is cochroach, but I swear I’ve seen beetle people use 4f11 instead of 4D9

Index of Other’s Results

Even SkippedBrown & Denell 1997

Choe & Brown 2006Grasshopper antigen, see also Patel 1994

Index of Other’s Results

NubbinLi & Popadic 2004 Evolution& Development

A monoclonal antibody that recognizes a conserved epitope in the Nub/pdm protein has been generated and used successfully in a number of crustacean and chelicerate species (Averof and Cohen 1997; Abzhanov and Kaufman 2000; Damen et al. 2002). Unfortunately, this antibody does not recognize insect nub orthologs except for that of Drosophila. Index of Other’s Results

Pairberry (Prd, Gsb, Gsbn)Davis & Patel 2001

Index of Other’s Results

Phospho-SMADNunes da fonseca & Roth 2008 Dev Cell

Index of Other’s Results

Phospho-TyrosineNunes da fonseca & Roth 2008 Dev Cell

Anti-phosphotyrosine antibody staining marks the cell outlines (red), DAPI (blue)

Index of Other’s Results

Synapsin 1Dreyer&Schactner2010

The monoclonal primary antibody from mouse against a fusion protein consisting of a glutathione-S-transferase and the first amino acids of the presynaptic vesicle protein synapsin I coded by its 5 -end ′(SYNORF1, Klagges et al., 1996 )

Index of Other’s Results