Monoclonal 3G8- pronephros and otic vesicle

Generated and characterized by Elizabeth Jones [1]. References: Dev Bio 171:531- [2]

Antibody name: 3G8

Raised in: mouse, monoclonal

Subtype: IgG

Antigen: dissected pronephroi. Specific target unknown.

Developmental Window: First detects target at stage 31, continues until latest stages tested. As this antibody stained only the lumen of the pronephric proximal tubules the shape of the organ looks odd in early stages and only begins to look like a pronephros at around stage 34 when a well luminized epithelial tubule network has formed.

Stains: proximal pronephric nephron, nephrostomes and otic vesicle,

Fixatives: formalin, MEMFA, TCA are all OK. Following fixation sample must be dehydrated with methanol for the antibody to work optimally. Others not tested. The target degrades over time in alcohol, so samples should be stored for less than one month post-fixation.

Sensitive to bleaching: No, though store time in methanol may be shortened by bleaching

Immunoprecipitation/Western blotting: i.p unknown, does not work on Westerns.

Available as: cell supernatent, 3 x

Available from; Liz Jones (ea@dna.bio.warwick.ac.uk) , 10 ml 3x. Sent on dry ice so please provide account number for shipping costs.

Optimized conditions: Very clean antibody, very stable, can be reused. Development in BCIP/NBT usually takes 30 minutes, in red, about 60 minutes. Works well in immunofluorescense. As the pronephros is only covered by a thin transparent epidermis it is not necessary to clear stained embryos. In cleared embryos both pronephroi are often in view, one in focus and one blurred, and this can make photography difficult see for example stage 32 sample above.

Other species: works on other anurans (Bombina), and also on axolotls when formalin plus 10% DMSO is used as the fixative, but it isn't nearly as clean as with Xenopus or Bombina. This is probably just a protocol problem. Reported to work on zebrafish.

XAO:

References: Dev Bio 171:531- [3]