Rhodamine conjugated chitin binding probe

Manufactured by New England Biolabs

The Rhodamine-conjugated Chitin-Binding Probe is a fluorescent probe designed to detect the presence of chitin, a polysaccharide found in the cell walls of fungi and the exoskeletons of arthropods. The probe consists of a rhodamine fluorescent dye conjugated to a chitin-binding domain, allowing it to bind to and visualize chitin structures.

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Lab products found in correlation

4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (2 mentions) Mouse anti 2a12, by Developmental Studies Hybridoma Bank (1 mentions) Alexa fluor 488, by Thermo Fisher Scientific (1 mentions) Cyanine3 (cy3), by Jackson ImmunoResearch (1 mentions)

2 protocols using rhodamine conjugated chitin binding probe

Fixation and Immunostaining of Drosophila Embryos

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Drosophila embryos were formaldehyde-fixed and processed as described previously (Tepass et al., 1990 (link)). For Arm staining, embryos were heat-fixed (Gamblin et al., 2014 (link)). Primary antibodies used: mouse anti-2A12 (1:10 dilution; Developmental Studies Hybridoma Bank, DSHB), mouse anti-Cora (1:500; clones C566.9 and C615.16, DHSB), mouse and anti-Tango (1:10; DHSB), mouse anti-Arm (1:250; N2-7A1, DHSB), mouse anti-Fas3 (1:10; 7G10, DHSB), rabbit anti-Verm (1:500; Luschnig et al., 2006 (link)), rabbit anti-Serp (1:500; Luschnig et al., 2006 (link)), rabbit anti-aPKC (1:250; C-20, Santa Cruz Biotechnology) and rat anti-Crb (1:500; this study). Secondary antibodies were conjugated to Cy3 (Jackson Immunoresearch Laboratories) or Alexa Fluor 488 (Molecular Probes). Rhodamine-conjugated Chitin-Binding Probe (New England BioLabs) was used at a concentration of 4 µg/ml, and co-incubated with secondary antibodies.

Sollier K., Gaudé H.M., Chartier F.J, & Laprise P. (2015). Rac1 controls epithelial tube length through the apical secretion and polarity pathways. Biology Open, 5(1), 49-54.

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Immunofluorescence Staining of Embryos

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For immunofluorescence experiments, embryos were dissected on subbing solution-coated slides (4 mg/ml gelatin USP, 0.4 mg/ml chromalum, 1 mg/ml poly-L-lysine) in 0.75x egg salts (1x egg salts: 118 mM NaCl, 40 mM KCl, 3.4 mM MgCl₂, 3.4 mM CaCl₂, 5 mM Hepes pH7.4), freeze cracked, and fixed in −20°C methanol for 15 minutes as described previously (Olson et al., 2012 (link)). Following rehydration in PBS, slides were stained overnight with a 1:100 dilution of rhodamine-conjugated chitin-binding probe (New England Biolabs, discontinued) and 1 ug/ml DAPI (ThermoFisher, D1306) before being mounted in 0.5% p-phenylenediamine in 90% glycerol, 20 mM Tris pH 8.8. In some cases, the eggshell vitelline layer was removed prior to the freeze-cracking step by brief alkaline-bleach treatment (0.5 N NaOH, 2.5% bleach in M9 buffer). In all cases, a single central plane was imaged.

González D.P., Lamb H.V., Partida D., Wilson Z.T., Harrison M.C., Prieto J.A., Moresco J.J., Diedrich J.K., Yates JR I.I.I, & Olson S.K. (2018). CBD-1 organizes two independent complexes required for eggshell vitelline layer formation and egg activation in C. elegans. Developmental biology, 442(2), 288-300.

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