2-4 h embryos were fixed as previously described75 (link) and stored in methanol at −20°C until required. Fixed embryos were placed in Wheaton vials (Sigma, Cat# Z115053-12EA) for FISH as described previously.31 (link) Embryos were probed for the mRNA target using smiFISH fluorescent probes designed to exonic sequences of gt, sog and mew with X or Z flap sequences70 (link) and secondary detection probes labeled with Quasar 570 or 670 fluorophore (all probe sequences are listed in Table S1 ). Mouse α-Spectrin antibody (1:50 DSHB, 3A9 (323 or M10-2), RRID:AB_528473 ) incubation overnight at 4°C was used with a secondary Goat anti-Mouse IgG (H + L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 488 (1:1000 Thermo Fisher Scientific, Cat# A-11001, RRID:AB_2534069 ) for 2 h at room temperature to stain the membrane. DAPI (New England Biolabs, Cat# 4083) was added to the embryos in the second of the final four washes of the protocol at a concentration of 1:1000 and embryos were mounted onto slides in Prolong Diamond (Thermo Fisher Scientific, Cat# P36961) to set overnight before imaging.
4 6 diamidino 2 phenylindole (dapi)
Manufactured by New England Biolabs
DAPI (4',6-diamidino-2-phenylindole) is a fluorescent stain that binds to the minor groove of DNA. It is commonly used in fluorescence microscopy and flow cytometry to visualize and quantify nucleic acids.
Automatically generated - may contain errors
Lab products found in correlation
Prolong diamond antifade mountant, by Thermo Fisher Scientific (3 mentions)
Prolong diamond, by Thermo Fisher Scientific (1 mentions)
Wheaton vials, by Merck Group (1 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 647, by Thermo Fisher Scientific (1 mentions)
Alex fluor 488, by Thermo Fisher Scientific (1 mentions)
Mouse anti biotin, by Roche (1 mentions)
4 protocols using 4 6 diamidino 2 phenylindole (dapi)
1
Fluorescence In Situ Hybridization of Drosophila Embryos
2
Spatiotemporal Transcriptomic Analysis of Drosophila Embryo
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For smFISH, 2–4 h or 1–3 h embryos were processed as described (Hoppe et al., 2020 (link)) with anti-sog Stellaris (Fig. 2 C, Fig. S1 ), or sog single molecule inexpensive FISH (smiFISH) (Fig. S4 ) (Tsanov et al., 2016 (link)), ush Stellaris, lacZ Stellaris, and Race smiFISH probes (Tsanov et al., 2016 (link)). ush probe sequences are available from (Hoppe et al., 2020 (link)), while sog Stellaris, sog smiFISH, lacZ Stellaris, and Race smiFISH probe sequences are provided in Table S2 . Race and sog smiFISH probes were annealed to a 570-conjugated Y-FLAP and Z-FLAP, respectively (Tsanov et al., 2016 (link)). For immunostaining against mNeonGreen and Spectrin, mouse anti-mNeonGreen [32F6] (1:500, ChromoTek, cat. #32f6-100, RRID: AB_2827566), mouse anti-Spectrin (1:50, DSHB, cat. #3A9 (323 or M10-2), RRID: AB_528473) and donkey anti-mouse IgG secondary antibody, Alexa Fluor 488 (1:1000, Thermo Fisher Scientific, cat. #A-21202, RRID: AB_141607) primary and secondary antibodies were used. To stain nuclei, samples were incubated with DAPI (1:1000, NEB 4083). Samples were mounted in ProLong™ Diamond Antifade Mountant (Thermo Fisher Scientific, P36961).
3
Comprehensive Embryonic RNA Staining
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Embryos (2–4 h) were collected and stained by RNA in situ hybridisation with sog-digoxygenin-UTP, Race-Biotin-UTP, lacZ-digoxygenin-UTP or mNeonGreen-biotin-UTP probes as described (Hoppe et al., 2020 (link); Kosman et al., 2004 (link)). An mNeonGreen-biotin-UTP probe was synthesised as previously described (Kosman et al., 2004 (link)) with primers listed in Table S1 . Antibodies used were mouse anti-biotin (1:250, Roche, cat. #1297597), sheep anti-digoxigenin Fab fragments antibody, AP conjugated (1:200, Roche, cat. #11093274910 RRID:AB514497), donkey anti-mouse IgG secondary antibody, Alexa Fluor 647 (1:500, Thermo Fisher Scientific, cat. #A-31571, RRID:AB162542), and donkey anti-sheep IgG secondary antibody, Alex Fluor 488 (1:500, Thermo Fisher Scientific, cat. #A-11015, RRID: AB_2534082). For pMad immunostaining, anti-Smad3 (phospho S423+S425) [EP823Y] (1:500, Abcam, cat. #ab52903, RRID: AB_882596) primary antibody and Donkey anti-rabbit IgG secondary antibody, Alexa Fluor 647 (1:500, Thermo Fisher Scientific, cat. #A-31573, RRID: AB_2536183) were used. To stain embryo nuclei, samples were incubated with DAPI (1:1000, NEB 4083). Samples were mounted in ProLong™ Diamond Antifade Mountant (Thermo Fisher Scientific, P36961).
4
Embryonic in situ Hybridization and Antibody Staining
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Embryo collections (2-4h), RNA probe synthesis and in situ hybridization with digoxygenin-UTP-labeled (Sigma, 11277073910) or biotin-UTP-labeled probes (Sigma, 11685597910) were performed as described by (Kosman et al., 2004 (link)). Antisense probes targeting sog and brk gene regions were approximately 1kb in length (Primer sequences available upon request). The following primary and secondary antibodies were used: Sheep Anti-Digoxigenin Fab fragments Antibody, AP Conjugated (1:250 Roche Cat# 11093274910, RRID:AB_514497 ), mouse anti-biotin (1:250 Roche, 1297597), donkey anti-Sheep IgG Secondary Antibody, Alexa Fluor or 555 (1:500; Thermo Fisher Scientific Cat# A-21436, RRID:AB_2535857 ) and donkey anti-Mouse IgG Secondary Antibody, Alexa Fluor 647 (1:500; Thermo Fisher Scientific Cat# A-31571, RRID:AB_162542 ). Samples were incubated with DAPI (1:500; NEB, 4083) and mounted in ProLongTM Diamond Antifade Mountant (Thermo Fisher, P36961). For alkaline phosphatase blue stain; embryos were incubated in staining solution containing 0.675mg/mL nitro blue tetrazolium (NBT Sigma Cat# 11585029001) and 0.35mg/mL 5-bromo-4-chloro-3-indolyl-phosphate (BCIP Sigma Cat# B6149). Embryos were mounted in PermountTM (BioWORLD Cat# 21750009).
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!