Rat anti de cad

Manufactured by Developmental Studies Hybridoma Bank

Sourced in Germany

Rat anti-DE-Cad is a monoclonal antibody produced by the Developmental Studies Hybridoma Bank. It is designed to detect the Drosophila E-cadherin (DE-Cad) protein, which is involved in cell-cell adhesion in Drosophila melanogaster.

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4 protocols using rat anti de cad

Immunohistochemical Procedure for Drosophila Larvae

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Larvae were fixed in 3.7% formaldehyde at room temperature for 20 min, after which wing imaginal discs were dissected. The following primary antibodies were used: rat anti-DE-Cad (1:50) (Developmental Studies Hybridoma Bank (DSHB)), mouse anti-GFP (1: 5,000 for Western blotting, Millipore), rabbit anti-Myc (1:100, Santa Cruz Biotechnology), mouse anti-β-Galaxtosidase (1:500, Promega), mouse anti-human YAP (1:50), rabbit anti-cleaved-Dcp-1 (1:200, Cell Signaling Technology), mouse anti-human α-Cat (1:50, Santa Cruz Biotechnology), rabbit anti-human Scrib (1:50, Abcam), and rabbit anti-human YAP (1:50, Cell Signaling Technologies).
Secondary antibodies (1:200) were as follows: goat anti-mouse IgG Alexa 488, goat anti-mouse IgG Alexa 568, goat anti-mouse IgG Alexa 647, goat anti-rabbit IgG Alexa 488, goat anti-rabbit IgG Alexa 568, goat anti-rat IgG Alexa 488 and Alexa488-conjugated phalloidin (all from Thermo Fisher Scientific).

Huang Y., Gui J., Myllymäki S.M., Roy K., Tõnissoo T., Mikkola M.L, & Shimmi O. (2022). Scribble and α-Catenin cooperatively regulate epithelial homeostasis and growth. Frontiers in Cell and Developmental Biology, 10, 912001.

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Pupal Eye Immunostaining Protocol

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Pupal eyes or wing imaginal discs were fixed and stained following standard formaldehyde fixation and permeabilization/washes in PBS containing 0.3% Triton X-100. All pupal eyes were fixed ∼40 h APF unless otherwise specified. For phalloidin staining after Triton X-100 permeabilization, tissue was fixed first, incubated in PBS containing 0.3% Triton X-100 and 5% goat serum for 1 h, and then washed three times in PBS with 5% goat serum and stained with phalloidin in PBS with 5% goat serum for 30 min. The following antibodies were used: rat anti–DE-cad (1:10), mouse anti–α-Spec (1:50), mouse anti-Dlg (1:50), mouse anti-Ena (1:50; all from the Developmental Studies Hybridoma Bank), mouse anti-Myc (1:200, clone 9E10; Millipore Sigma), rabbit anti-pMLC (1:10; Cell Signaling Technologies), rabbit anti–PAR-3 (1:1,000; provided by A. Wodarz, Univesity of Cologne, Cologne, Germany; Wodarz et al., 1999 (link)). Alexa Fluor 568 phalloidin (1:50; Invitrogen) was used to visualize F-actin.
In experiments involving treatment of Rok inhibitor, pupal eyes were incubated with 1 mM Y27632 in Schneider’s Drosophila medium (GIBCO BRL, Life Technologies) for 1 h before fixation as described previously (Legoff et al., 2013 (link)).

Deng H., Yang L., Wen P., Lei H., Blount P, & Pan D. (2020). Spectrin couples cell shape, cortical tension, and Hippo signaling in retinal epithelial morphogenesis. The Journal of Cell Biology, 219(4), e201907018.

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Drosophila Ovary Immunostaining Protocol

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1–2 day old females were incubated at 29°C for 1–2 days in vials with fresh yeast paste and males. Ovaries were dissected, fixed in 4% paraformaldehyde for 20 mins in PBS with 0.1% Triton-X (PBST), washed 5 times with PBST and blocked in normal goat serum (NGS) for 30 mins. For primary antibody staining, ovaries were incubated with rat anti-DE-cad (1:100, Developmental Studies Hybridoma Bank) and rabbit anti-GFP (1:100, Thermo Fisher Scientific) overnight at 4°C and washed 10 times the next day over 1 hour. Next, ovaries were incubated with Alexa Fluor dye labeled secondary antibodies (1:150, Thermo Fisher Scientific) and Alexa Fluor dye conjugated phalloidin (1:150, Thermo Fisher Scientific) for 2 hours. Egg chambers were then incubated in DAPI for 10 mins. Ovaries were washed 5 times in PBST and washed overnight and washed again 5 times next morning. Ovaries were stored and mounted on microscope slide in 70% glycerol and then imaged on a Nikon A1 scanning confocal microscope. In Fig 5, image z-stacks were first deconvolved (3D Deconvolution) in NIS-Elements (Nikon) and assembled into maximum projections for presentation.

Raza Q., Choi J.Y., Li Y., O’Dowd R.M., Watkins S.C., Chikina M., Hong Y., Clark N.L, & Kwiatkowski A.V. (2019). Evolutionary rate covariation analysis of E-cadherin identifies Raskol as a regulator of cell adhesion and actin dynamics in Drosophila. PLoS Genetics, 15(2), e1007720.

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Immunostaining and in situ Hybridization in Drosophila

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We followed standard protocols for immunostainings and in situ hybridisations. Embryos were staged as described [42 ]. Imaginal discs were obtained by dissecting third instar larvae.
The following primary antibodies and dilutions were used: mouse anti-2A12 (recognises Gasp, 1:10), rat anti-DEcad (1:100), and mouse anti-Crb (1:20) from Developmental Studies Hybridoma Bank, DSHB; rbb anti-Verm (1:300) from S. Luschnig; goat anti-GFP (1:600) Molecular Probes and Roche; ck anti-ßGal (1:500) abCAM; GP anti-Uif (1:400) from R. Ward; and rbb anti-Pio (1:100) from M. Affolter. CBP (chitin-binding probe) conjugated with Cy3, Cy2 and Cy5 was used at 1:300 (generated by N. Martin). WGA conjugated with Alexa-555, -488, and -647 was used at 1:300 (Molecular Probes). Cy3-, Cy2- and Cy5-conjugated secondary antibodies (Jackson ImmunoResearch) were used at 1:300.
A reb riboprobe was generated using the following primers:

Forward: 5′- AACTGTGCCTCGGCGCTAGTC

Reverse: 5′- AGCAGTCGAAACACGCAGCTT

Confocal images were acquired with a Leica TCS-SPE system. Images were post-processed with ImageJ and Adobe Photoshop and assembled using Adobe Illustrator.

Moussian B., Letizia A., Martínez-Corrales G., Rotstein B., Casali A, & Llimargas M. (2015). Deciphering the Genetic Programme Triggering Timely and Spatially-Regulated Chitin Deposition. PLoS Genetics, 11(1), e1004939.

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