Whole fish in situ hybridization was performed as described previously.6 (link) Fluorescence in situ hybridization and immunofluorescence were performed with an RNAscope kit from Advanced Cell Diagnostics, Inc, following the protocol described by Gross-Thebing et al.18 (link) Fluorescence in situ hybridization probes to detect tie1 mRNA and tie1AS lncRNA were designed by Advanced Cell Diagnostics, Inc. Probe details are mentioned in the Major Resource Table in the online-only Data Supplement . Anti-Elav antibody (Invitrogen; A21271) was used as the primary antibody to detect Elavl1, and Alexa Fluor 488 goat anti-mouse antibody (Invitrogen A11001) was used as the secondary. Control immunofluorescence staining was performed with anti-mouse IgG (Cell Signaling; 7076) as the primary antibody and Alexa Fluor 488 goat anti-mouse antibody (Invitrogen A11001) as the secondary.
Protocol detail
In Situ Hybridization and Immunofluorescence of tie1 and tie1AS
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Alexa fluor 488
Anti antibody
Anti igg
Antibody
Cell
Diagnostics
Fish
Fluorescence in situ hybridization
Goat
Immunofluorescence
In situ hybridization
Lncrna
Mouse
Mrna
Supplement
Corresponding Organization :
Other organizations : Massachusetts General Hospital, Harvard University, Medical College of Wisconsin
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Variable analysis
independent variables
- Whole fish in situ hybridization
- Fluorescence in situ hybridization
- Immunofluorescence
- Detection of tie1 mRNA and tie1AS lncRNA expression
- Anti-mouse IgG as the primary antibody for control immunofluorescence staining
- Positive control: Anti-Elav antibody to detect Elavl1
- Negative control: Anti-mouse IgG as the primary antibody for control immunofluorescence staining
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