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Donkey fab fragments

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Donkey Fab fragments are antigen-binding fragments derived from donkey antibodies. They are prepared by enzymatic digestion of whole donkey antibodies. Donkey Fab fragments retain the antigen-binding capacity of the parent antibody while being smaller in size.

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Lab products found in correlation

Lsm 700 confocal microscope, by Zeiss (5 mentions) Cleaved caspase 3 cc3, by Cell Signaling Technology (3 mentions) Zen 2012 imaging software, by Zeiss (3 mentions) β galactosidase, by Merck Group (2 mentions) Axio imager, by Zeiss (2 mentions) Anti cleaved caspase 3, by Cell Signaling Technology (1 mentions) β gal, by Promega (1 mentions) Prism 7, by GraphPad (1 mentions) H2dcfda, by Thermo Fisher Scientific (1 mentions) Cleaved caspase 3, by Cell Signaling Technology (1 mentions) Tunel assay kit, by Roche (1 mentions)

6 protocols using donkey fab fragments

1

Late Larval Imaginal Disc Immunostaining

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Imaginal discs were dissected from late 3rd instar larvae and stained using standard protocols. Antibodies to the following primary antigens were used: PH3 (Upstate), anti-cleaved Caspase-3 (Cell Signaling), β-GAL (Promega), ELAV and Wg (DHSB). Secondary antibodies were donkey Fab fragments from Jackson ImmunoResearch. Images were taken with either a Zeiss AxioImager or a confocal microscope.
Fan Y., Wang S., Hernandez J., Yenigun V.B., Hertlein G., Fogarty C.E., Lindblad J.L, & Bergmann A. (2014). Genetic Models of Apoptosis-Induced Proliferation Decipher Activation of JNK and Identify a Requirement of EGFR Signaling for Tissue Regenerative Responses in Drosophila. PLoS Genetics, 10(1), e1004131.
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2

Midgut Dissection and Immunostaining

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Intact female midguts were dissected using standard protocols (Amcheslavsky et al., 2009 (link)). Primary antibodies were: PH3 (Millipore: 1:2,000), β-Galactosidase (DSHB; 1:100 (concentrate)), NimC (Kurucz et al., 2007 (link); 1:300); Dronc (SK11) (Wilson et al., 2002 (link); 1:200); cleaved caspase 3 (CC3) (Cell Signaling Technology; 1:400). Secondary antibodies were donkey Fab fragments from Jackson ImmunoResearch. If not noted otherwise, region R4ab in the posterior midgut was imaged and analyzed. Images were obtained with a Zeiss LSM 700 confocal microscope, analyzed with Zen 2012 imaging software (Carl Zeiss) and processed with Adobe Photoshop CS6.
Amcheslavsky A., Lindblad J.L, & Bergmann A. (2020). Transiently ‘‘Undead’’ Enterocytes Mediate Homeostatic Tissue Turnover in the Adult Drosophila Midgut. Cell reports, 33(8), 108408.
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3

Fluorescence Microscopy of Apoptosis Markers

Cited 2 times
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DHE and H2DCF-DA (both from Invitrogen/Molecular Probes) labeling of unfixed tissue was performed as described (Owusu-Ansah et al., 2008 (link)). TUNEL labeling (Roche) was done according to the manufacturer’s instructions. Antibody labelings were done on fixed tissue following standard procedures (Fan and Bergmann, 2014 (link); Fogarty and Bergmann, 2014 (link)). The following antibodies were used: cleaved caspase-3 (CC3; Cell Signaling Technology); NimC (kind gift of I. Andó) (Kurucz et al., 2007 (link)); MMP1 (Developmental Studies Hybridoma Bank (DSHB)) and pJNK (Promega). Secondary antibodies were donkey Fab fragments from Jackson Immunoresearch. Eye/antennal cephalic complexes were counterlabeled with the nuclear dye DAPI to visualize tissue outline. Images were taken with a Zeiss LSM700 confocal microscope. For quantification of confocal images, the ‘Record Measurement’ function of Photoshop was used. Clones were outlined and signal intensity determined. Multiple clones of five to ten imaginal discs per genotype obtained in three independent experiments were measured. Analysis and graph generation was done using GraphPad Prism 7.03. The statistical method and the P values are indicated in the figure legends.
Pérez E., Lindblad J.L, & Bergmann A. (2017). Tumor-promoting function of apoptotic caspases by an amplification loop involving ROS, macrophages and JNK in Drosophila. eLife, 6, e26747.
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4

Imaging of Cell Death in Imaginal Discs

Cited 1 time
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At least 20 imaginal discs per experiment were dissected from late third instar larvae and pupal retinae from 42-h-old pupae. They were fixed and stained using standard protocols (Fogarty and Bergmann, 2014 (link)). TUNEL was performed using a TUNEL assay kit (Roche Life Sciences) according to the manufacturer's instructions. Antibodies to the following primary antigens were used: ELAV [rat; 1:50; Developmental Studies Hybridoma Bank (DSHB)]; Cut (mouse; 1:50; DSHB); Dlg (rabbit; 1:100; from Kwang-Wook Choi, Korea Advanced Institute of Science and Technology, Daejeon, South Korea); MBNL1 (rabbit; 1:2000; from Charles Thornton, University of Rochester Medical Center, Rochester, NY, USA); cleaved Caspase 3 (rabbit; 1:200; Cell Signaling Technology, 9661); and eIF2α (P-Ser51) (rabbit; 1:100; Cell Signaling Technology, 3597). Secondary antibodies were donkey Fab fragments from Jackson ImmunoResearch (715-166-151, 711-096-152, 712-606-153, 711-166-152; all at 1:600). Nuclei were visualized by Hoechst and DAPI staining. Fluorescent images were taken with an Olympus Optical FV500 confocal microscope.
Yenigun V.B., Sirito M., Amcheslavky A., Czernuszewicz T., Colonques-Bellmunt J., García-Alcover I., Wojciechowska M., Bolduc C., Chen Z., López Castel A., Krahe R, & Bergmann A. (2017). (CCUG)n RNA toxicity in a Drosophila model of myotonic dystrophy type 2 (DM2) activates apoptosis. Disease Models & Mechanisms, 10(8), 993-1003.
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5

Midgut Dissection and Immunostaining

Cited 2 times
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Intact female midguts were dissected using standard protocols59 (link). Primary antibodies were: anti Dronc60 (link) (1:200; a kind gift of Pascal Meier); PH3 (1:2,000; Millipore), Prospero (Pros, 1:20; DSHB; Prospero (MR1A) was deposited to the DSHB by C.Q. Doe); Pdm-161 (link) (1:1,000; a kind gift of Yu Cai). DAPI was used to counterstain nuclei. Phalloidin labeling was used to assess the physical properties of the guts. Secondary antibodies were donkey Fab fragments from Jackson ImmunoResearch. If not noted otherwise, region R4ab35 (link),36 (link) in the posterior midgut was imaged and analysed. Images were obtained with a Zeiss LSM 700 confocal microscope, analysed with Zen 2012 imaging software (Carl Zeiss) and processed with Adobe Photoshop CS6.
Lindblad J.L., Tare M., Amcheslavsky A., Shields A, & Bergmann A. (2021). Non-apoptotic enteroblast-specific role of the initiator caspase Dronc for development and homeostasis of the Drosophila intestine. Scientific Reports, 11, 2645.
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6

Midgut Dissection and Immunostaining

Cited 1 time
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Intact female midguts were dissected using standard protocols (Amcheslavsky et al., 2009 (link)). Primary antibodies were: PH3 (Millipore: 1:2,000), β-Galactosidase (DSHB; 1:100 (concentrate)), NimC (Kurucz et al., 2007 (link); 1:300); Dronc (SK11) (Wilson et al., 2002 (link); 1:200); cleaved caspase 3 (CC3) (Cell Signaling Technology; 1:400). Secondary antibodies were donkey Fab fragments from Jackson ImmunoResearch. If not noted otherwise, region R4ab in the posterior midgut was imaged and analyzed. Images were obtained with a Zeiss LSM 700 confocal microscope, analyzed with Zen 2012 imaging software (Carl Zeiss) and processed with Adobe Photoshop CS6.
Amcheslavsky A., Lindblad J.L, & Bergmann A. (2020). Transiently ‘‘Undead’’ Enterocytes Mediate Homeostatic Tissue Turnover in the Adult Drosophila Midgut. Cell reports, 33(8), 108408.
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