Mouse anti engrailed

Manufactured by Developmental Studies Hybridoma Bank

Sourced in Canada

Mouse anti-Engrailed is a monoclonal antibody that binds to the Engrailed protein, a transcription factor involved in embryonic development. This antibody can be used to detect and study the expression of Engrailed in various biological samples.

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5 protocols using mouse anti engrailed

Drosophila Ovary Immunohistochemistry

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The following primary antibodies were used: guinea-pig anti-Tj (G5 or GP6, 1:5000) [57 (link)], rat anti-Bab2 (R10, 1:3000; or R7, 1:2000) [20 (link)], rabbit anti-Vasa (1:2000) [80 (link)], rabbit anti-Vasa (d-260, 1:500; Santa Cruz Biotechnology), chicken anti-Vasa (1:5000; gift from K. Howard and M. Van Doren), rabbit anti-α-spectrin (#254, 1:1000; gift from D. Branton), mouse anti-LamC (LC28.26, 1:50), mouse anti-Hts (1B1, 1:5), mouse anti-N (C17.9C6, 1:5; C458.2H, 1:5), mouse anti-Dl (C594.9B, 1:5), mouse anti-Engrailed (4D9, 1:5), and mouse anti-ßPS integrin (CF.6G11, 1:10) (Developmental Studies Hybridoma Bank), rabbit anti-pMad (PS1, 1:250; gift from T. Tabata) [81 (link)], rabbit anti-pMad (pSmad1/5, 41D10, 1:100; Cell Signalling), rabbit anti-ß-galactosidase (1:1500; MP Biomedicals), and rabbit anti-GFP (1:100; BD Biosciences). Secondary antibodies (1:400) were conjugated either to Cy3, Cy5 (Jackson Immuno Research Laboratories), Alexa-405, Alexa-555, Alexa-488, or Alexa-647 (Molecular Probes, Life Technologies). Ovaries were mounted in Vectashield (Vector Laboratories).

Panchal T., Chen X., Alchits E., Oh Y., Poon J., Kouptsova J., Laski F.A, & Godt D. (2017). Specification and spatial arrangement of cells in the germline stem cell niche of the Drosophila ovary depend on the Maf transcription factor Traffic jam. PLoS Genetics, 13(5), e1006790.

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Immunofluorescent Protein Localization Protocol

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Immunofluorescent studies were performed using standard procedures (König and Shcherbata, 2013). Primary antibodies used were: guinea pig anti-Tj (1:10 000; a kind gift from Dorothea Godt, University of Toronto, Canada), rabbit anti-Vasa (1:5000; a gift from Herbert Jäckle, MPI-bpc, Göttingen), mouse anti-Engrailed (1:20; Developmental Studies Hybridoma Bank, DSHB), mouse anti-Fas3 (1:20; DSHB), rat anti-DE-Cadherin (1:20; DSHB), rabbit anti-mCherry (1:200; Abcam), and mouse anti-Flag (1:500; Sigma Aldrich). The following secondary antibodies were used: goat anti-guinea pig Alexa 647 (1:500; Life Technologies, A-21450), goat anti-mouse Alexa 488 (1:500; Molecular Probes) and goat anti-rabbit Alexa 568 (1:500; Molecular Probes). DAPI (Sigma) was used to stain the nuclei. Samples were imaged using Zeiss LSM 700. ImageJ and CorelDrawX8 were used to make Figures.

Karki P., Carney T.D., Maracci C., Yatsenko A.S., Shcherbata H.R, & Rodnina M.V. (2021). Tissue-specific regulation of translational readthrough tunes functions of the traffic jam transcription factor. Nucleic Acids Research, 50(11), 6001-6019.

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Immunohistochemical Analysis of Drosophila Larvae

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Larvae were dissected in cold phosphate buffered saline (PBS) and fixed in 4% formaldehyde in PBS for 30 min at room temperature. Larvae were then washed three times for 20 min with PBT and blocked in 2% normal donkey serum in PBT for 30 min. We incubated the tissue overnight at 4° in a primary antibody solution containing mouse anti-Engrailed (Developmental Studies Hybridoma Bank 4D9, 1:40) diluted in 2% normal donkey serum in PBT. After washing three times for 20 min in PBT, larvae were incubated in the dark with goat anti-mouse Alexa 568 (Invitrogen, 1:200) and TRICT-Phalloidin (Sigma, 1:200) diluted in 2% normal donkey serum in PBT overnight at 4°. Larvae were rinsed with PBT, and ovaries were mounted on a poly-l-lysine-coated coverslip using Fluoromount-G (SouthernBiotech).

Mendes C.C, & Mirth C.K. (2015). Stage-Specific Plasticity in Ovary Size Is Regulated by Insulin/Insulin-Like Growth Factor and Ecdysone Signaling in Drosophila. Genetics, 202(2), 703-719.

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Immunofluorescent Imaging of Drosophila Tissues

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Immunofluorescent studies were performed using standard procedures (König and Shcherbata, 2013) . Primary antibodies used were: guinea pig anti-Tj (1:10,000; a kind gift from Dorothea Godt, University of Toronto, Canada), rabbit anti-Vasa (1:5,000; a gift from Herbert Jäckle, MPI-bpc, Göttingen), mouse anti-Engrailed (1:20; Developmental Studies Hybridoma Bank, DSHB), mouse anti-Fas3 (1:20; DSHB), rat anti-DE-Cadherin (1:20; DSHB), rabbit anti-mCherry (1:200; Abcam), and mouse anti-Flag (1:500; Sigma Aldrich). The following secondary antibodies were used: goat anti-guinea pig Alexa 647 (1:500; Life Technologies, A-21450), goat anti-mouse Alexa 488 (1:500;
Molecular Probes) and goat anti-rabbit Alexa 568 (1:500; Molecular Probes). DAPI (Sigma) was used to stain the nuclei. Samples were imaged using Zeiss LSM 700. ImageJ and CoreDrawX8
were used to make figures.

Karki P., Carney T.D., Maracci C., Yatsenko A.S., Shcherbata H.R., & Rodnina M.V. (2020). Tissue-Specific Regulation of Translational Readthrough Tunes Functions of the Traffic Jam Transcription Factor.

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Immunofluorescence Staining of Drosophila Wing Discs

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Discs were dissected from wandering third instar larvae and immunofluorescence staining was carried out as previously described [33 (link)]. Briefly, larvae are dissected in cold PBS (pH7.4) and fixed in 4% formaldehyde in PBS for 15 minutes. Samples were then permeabilized in PBS + 0.1% Triton X-100 and blocked in 5% normal horse serum. Primary antibodies in 5% normal horse serum were incubated overnight at 4°C or 2 hours at RT. Primary antibodies used for the immunostainings were mouse or rabbit α-V5 (Life Technology), rabbit anti-pMad (1:200) (Cell Signaling), rabbit α-Spalt major (1:100), anti-hedgehog (1:40), and chicken α-lacZ (1:100). Mouse anti-patched (DSHB), mouse anti-engrailed (DSHB), rat anti-Ci 2A1 (DSHB) were obtained from Developmental Studies Hybridoma Bank (DSHB), University of Iowa, Department of Biological Sciences. Fluorophore conjugated secondary antibodies were from Jackson Immunoresearch. Confocal images were collected as single frames on a Zeiss LSM 780 confocal microscope. MG132 (100 μM; Calbiochem) in M3 medium (Sigma) was used to treat wing discs for up to 6 hours before immunostaining.

Wang G., Tang X., Chen Y., Cao J., Huang Q., Ling X., Ren W., Liu S., Wu Y., Ray L, & Lin X. (2014). Hyperplastic Discs Differentially Regulates the Transcriptional Outputs of Hedgehog Signaling. Mechanisms of development, 133, 117-125.

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