I would like to know what role each reagents (such as Prot k; PBST; PBS; Primary antibody; secondary antibody) play in the acylated tubulin method. And also can any one share the correct protocol to stain zebrafish brains at day 7 larvae
Acetylated tubulin (Ac-Tub) is more stabilized form of tubulin, resulting from post-translational modification of tubulin molecule. Ac-Tub antibody can recognize only Ac-Tub antigen within the cell. Stable components of centrosomes and microtubules (i.e of spindle fibers, neuronal microtubules). Therefore, Ac-Tub antibody is also used in tracking nuronal cells.
Proteinase K: break tough tissue covering the body to help more access and escape in washing to huge anitibody molecules.
-PBS: Phosphate buffered saline, pH ca 7.0~7.2
-PBST: PBS + 0.02% (v/v) + Tween 20 washing solution having neutral detergent
-Primary antibody (1st Ab): detecting and binding to Ac-Tub
-Secondary antibody (2nd Ab): detecting and binding to 1st Ab
(so, you know the site of Ac-Tub by 2nd Ab, so-called indirect immunofluorescent staining); 2nd Ab is usually conjugated to fluorescent dye [called FITC (green), TRITC (red) etc]. often conjugated to an enzyme to be detected via the signal precipitated by color development reaction.
There are links for your references.
-Zebrafish whole mount immunohistochemistry (https://www.youtube.com/watch?v=RDvo5V9xZxQ)
- PROTOCOL 012: ZEBRAFISH WHOLE MOUNT IMMUNOHISTOCHEMISTRY (Google)
Since the 7 day-old fry are very tough, need lots of washings, I guess.
Good luck.
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