Immunostaining and IF/FISH were carried out as described previously (Zimmerman et al. 2013 (link)). Primary antibodies used were: mouse anti-Broad core (Developmental Studies Hybridoma Bank [DSHB] 25E9.D7-concentrate, 1:500); rat anti-DE-cadherin (DSHB DCAD2- concentrate, 1:50); mouse anti-Gurken (DSHB 1D12-concentrate, 1:200); rabbit anti-Capicua (Kim et al. 2011 (link), 1:2000 in D. melanogaster; 1:500 in S. lebanonensis); rabbit anti-P-Mad (a gift from T. Jessell’s lab; 1:2000 in both species); rabbit anti-dpERK (Cell Signaling, 1:100). Alexafluor 488-, 568-, and 647-conjugated secondary antibodies (Molecular Probes/Invitrogen) were used at dilutions between 1:200 and 1:500. DAPI was used at a concentration of 1 αg/ml. Probes for FISH were diluted 1:500 in HYB mix.
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Immunostaining and IF/FISH Protocol
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Variable analysis
independent variables
- Primary antibodies used: mouse anti-Broad core, rat anti-DE-cadherin, mouse anti-Gurken, rabbit anti-Capicua, rabbit anti-P-Mad, rabbit anti-dpERK
- Outcomes of immunostaining and IF/FISH experiments
- Developmental Studies Hybridoma Bank (DSHB) antibodies used at specific dilutions
- Alexafluor 488-, 568-, and 647-conjugated secondary antibodies used at specific dilutions
- DAPI used at a concentration of 1 μg/ml
- FISH probes diluted 1:500 in HYB mix
- None specified
- None specified
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