Fixed embryos were analyzed in Figs. 7 , S1 B, S2 (C and D), and S3 (D and E). All other images shown in this study are of living embryos. To visualize α-catenin and armadillo/β-catenin, embryos were boiled for 10 s in 0.03% Triton X-100/0.4% NaCl and devitellinized in heptane/methanol. To visualize Myo-GFP and E-cadherin, embryos were fixed for 10 min in a 1:1 mixture of 37% FA/0.1 M phosphate buffer, pH 7.2, and hand devitellinized in 0.1 M phosphate buffer. Antibodies used were anti–α-catenin (1:50; Developmental Studies Hybridoma Bank), rabbit anti–armadillo/β-catenin (1:100, made by J.A. Zallen as described by Riggleman et al., 1990 (link)), rat anti–E-cadherin (1:50; Developmental Studies Hybridoma Bank), and rabbit anti-GFP (1:150; Torrey Pines). Secondary antibodies conjugated to Alexa Fluor 488, 546, or 647 fluorophores (Molecular Probes) were used at a concentration of 1:500. Embryos were mounted in ProLong Gold (Invitrogen) between two coverslips for imaging. Images were acquired on a Zeiss LSM 700 confocal microscope with a PlanNeo 40×/1.3-NA oil-immersion objective (1.1-µm optical section and 0.56-µm z-steps).
Anti α catenin
Manufactured by Developmental Studies Hybridoma Bank
Anti–α-catenin is a laboratory reagent used for the detection and quantification of α-catenin, a protein involved in cell-cell adhesion. It functions as a primary antibody in various immunoassays and techniques, such as Western blotting, immunohistochemistry, and immunocytochemistry.
Automatically generated - may contain errors
Lab products found in correlation
Lsm 700 confocal microscope, by Zeiss (2 mentions)
Rat anti e cadherin, by Developmental Studies Hybridoma Bank (1 mentions)
Prolong gold, by Thermo Fisher Scientific (1 mentions)
Sp8 laser scanning confocal microscope, by Leica (1 mentions)
Axiophot microscope, by Zeiss (1 mentions)
Imagej, by Adobe (1 mentions)
2 protocols using anti α catenin
1
Imaging Embryos with Antibody Staining
2
Embryonic Cuticle and Antibody Staining
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To prepare embryonic cuticle, embryos were de-chorionated in 50% bleach for 5 min, washed with 0.1% TritonX-100, mounted in 1:1 solution of Hoyer’s and lactic acid and incubated overnight at 60 °C. Embryos were examined under phase contrast using a Zeiss Axiophot microscope.
The heat fixation method78 (link) was used for antibody staining of α-Cat1 embryos expressing UAS-driven transgenes with da-Gal4. Embryos over-expressing transgenes using Paired-Gal4 were fixed in a 1:1 mixture of 5% formaldehyde in 1× PBS and Heptane for 20 m. In both cases, embryos were devitellinized using a 1:3 mixture of Heptane and Methanol. Primary antibodies used were: rat mAb anti-HA (3F10, 1:500, Sigma), guinea pig pAb anti-α-Catenin (p121; 1:1000, Sarpal et al., 2012), mouse mAb anti-Arm (N2-7A1, 1:50; Developmental Studies Hybridoma Bank [DSHB]). Fluorescent secondary antibodies were used at a dilution of 1:400 (Jackson ImmunoResearch Laboratories and Invitrogen). Samples were analyzed on a Leica SP8 scanning laser confocal microscope using a 40x oil immersion lens (NA 1.3). Images were prepared and assembled in ImageJ, Adobe Photoshop, and Adobe Illustrator.
The heat fixation method78 (link) was used for antibody staining of α-Cat1 embryos expressing UAS-driven transgenes with da-Gal4. Embryos over-expressing transgenes using Paired-Gal4 were fixed in a 1:1 mixture of 5% formaldehyde in 1× PBS and Heptane for 20 m. In both cases, embryos were devitellinized using a 1:3 mixture of Heptane and Methanol. Primary antibodies used were: rat mAb anti-HA (3F10, 1:500, Sigma), guinea pig pAb anti-α-Catenin (p121; 1:1000, Sarpal et al., 2012), mouse mAb anti-Arm (N2-7A1, 1:50; Developmental Studies Hybridoma Bank [DSHB]). Fluorescent secondary antibodies were used at a dilution of 1:400 (Jackson ImmunoResearch Laboratories and Invitrogen). Samples were analyzed on a Leica SP8 scanning laser confocal microscope using a 40x oil immersion lens (NA 1.3). Images were prepared and assembled in ImageJ, Adobe Photoshop, and Adobe Illustrator.
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