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Biological Description
| Specificity | Deadpan Rat mAb detects endogenous levels of total Deadpan protein. |
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| Background | Deadpan (dpn) is a transcriptional repressor belonging to the Hairy/Enhancer of Split (HES) subclass of basic Helix-Loop-Helix (bHLH) proteins in Drosophila. It functions as a key regulator of neurogenesis and sex determination by promoting the self-renewal of neural stem cells (neuroblasts) and inhibiting their differentiation. Deadpan is regulated by the Notch signaling pathway and is expressed in neural cells as well as in other tissues like wing, eye, and leg imaginal discs. Structurally, Deadpan contains a bHLH domain that facilitates DNA binding and dimerization, an Orange domain involved in protein-protein interactions, and a WRPW motif essential for transcriptional repression. Deadpan's role in repressing specific gene transcription is crucial for the proper development of neural and other tissues in Drosophila. |
Usage Information
| Application | IHC | Dilution |
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|---|---|---|---|---|---|
| Reactivity | D. Melanogaster | ||||
| Source | Rat | MW | 47 kDa | ||
| Storage Buffer | PBS, pH 7.2+50% Glycerol+0.05% BSA+0.01% NaN₃ | Storage (from the date of receipt) |
–20°C (avoid freeze-thaw cycles), 2 years | ||
| IHC |
Experimental Protocol： Deparaffinization/Rehydration
1. Deparaffinize/hydrate sections:
2. Incubate sections in three washes of xylene for 5 min each.
3. Incubate sections in two washes of 100% ethanol for 10 min each.
4. Incubate sections in two washes of 95% ethanol for 10 min each.
5. Wash sections two times in dH2O for 5 min each.
6.Antigen retrieval: For Citrate: Heat slides in a microwave submersed in 1X citrate unmasking solution until boiling is initiated; continue with 10 min at a sub-boiling temperature (95°-98°C). Cool slides on bench top for 30 min.
Staining
1. Wash sections in dH2O three times for 5 min each.
2. Incubate sections in 3% hydrogen peroxide for 10 min.
3. Wash sections in dH2O two times for 5 min each.
4. Wash sections in wash buffer for 5 min.
5. Block each section with 100–400 µl of blocking solution for 1 hr at room temperature.
6. Remove blocking solution and add 100–400 µl primary antibody diluent in to each section. Incubate overnight at 4°C.
7. Remove antibody solution and wash sections with wash buffer three times for 5 min each.
8. Cover section with 1–3 drops HRPas needed. Incubate in a humidified chamber for 30 min at room temperature.
9. Wash sections three times with wash buffer for 5 min each.
10. Add DAB Chromogen Concentrate to DAB Diluent and mix well before use.
11. Apply 100–400 µl DAB to each section and monitor closely. 1–10 min generally provides an acceptable staining intensity.
12. Immerse slides in dH2O.
13. If desired, counterstain sections with hematoxylin.
14. Wash sections in dH2O two times for 5 min each.
15. Dehydrate sections: Incubate sections in 95% ethanol two times for 10 sec each; Repeat in 100% ethanol, incubating sections two times for 10 sec each; Repeat in xylene, incubating sections two times for 10 sec each.
16. Mount sections with coverslips and mounting medium.
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