Tor70; notochord

Monoclonal Tor70 notochord pattern

Notochord specific from late neurula to stage 28 (details below). Data from Richard Harland and Francesca Mariani.

Stage 22 embryo stained with Tor70 (brown) after in situ hybridization for En-2 (faint purple). Background is sometimes better on whole embryos that have been subjected to in situ hybridization before immunohistochemistry. This embryo was pigmented, and was bleached after the double staining.

Immunohistochemistry with Tor70 antibody

From Bolce, M.E. Hemmati-Brivanlou, A., Kushner, PD. and Harland, R.M. (1992) Ventral ectoderm of Xenopus forms neural tissue, including hindbrain, in response to activin. Development 115, 681-688

Tor 70 (Kushner, 1984) had previously been found to stain zebrafish notochord (P.D. Kushner and B. Mendelson, unpublished information). In Xenopus the antibody is specific to notochord from the late neurula stage to about stage 28. The notochord staining declines, as staining appears in the otic vesicle, cranial ganglia and a few proctodeal and stomodeal cells. At stage 40, staining is absent from the notochord, but prominent in lateral line, proctodeum, stomodeum, and nasal pits. We have not investigated the nature of the antigen in Xenopus further.

Tor 70 hybridoma cells

Mouse monoclonal probably IgM. Use an anti Ig secondary, such as that from Jackson or Amersham.
Derivation
Tor 70 hybridoma cells are the PEG-fusion product of NS1 x Balb/C spleen cells.
Antibody type
Original citation; P.D. Kushner, A library of monoclonal antibodies to Torpedo cholinergic synaptosomes. J. Neurochemistry 43: 775-786, l984.
Culture; See attached protocol developed by Ann Fischer.
Special notes:
a. NS1-derived hybridomas can be unstable; they may be best recloned upon thawing. The line being sent has been sub-cloned 4 successive times, three times by Pinky Kushner, one time by Ann Fisher.
b. NS1 cells can secrete a light chain (hence are sometimes called "silent secreters"). Screens for antibody production should therefore be specific, e.g., on tissue to be analyzed such as frog notochord or the original
immunogen; Torpedo presynaptic terminals from the electric organ.

Revised Tor70 growth protocol.

When grown in the medium given below, this cell line appears to be fairly stable. A frozen sample was tested and 10/10 clones made Tor70 antibody (though there was some variation in the quality in these small scale preps).

Tor 70 Antibody-producing lines: Protocol for growing

Medium:
500 ml Iscove's Modified Dulbecco's Medium (IMDM)
75 ml Fetal Bovine Serum (FBS)
5 ml Penicillin-Streptomycin (optional)

1. Rapidly thaw 1 vial frozen cells in 37 deg water bath.
2. Wash cells in 5ml complete medium.
3. Resuspend cells in 5ml complete medium. Cell density should be
0.5-1x106 cells/ml. Place into T-25 flask. Incubate at 37 degrees with 5% CO2.
4. Examine the cells the following day. If viable cells are about 1x106/ml, split 1/4. Cells are detached by gentle pipetting.
5. Cells are routinely split 1/4 to 1/6 twice a week. Supernatants are collected when medium becomes yellowish on day 4 or 5.

Stability of this antibody supernatant has been tested:
Fresh thawed.
Thawed, frozen, thawed (one cycle)
several days at 4 degrees C
several days room temp
All gave equivalent staining, optimum seems to be 1:5.

A fedex account number is preferred for us to send the cell line and antibody. Please let me know of any difficulty or even success with growing the line. Our batches of supernatant have not been quite as good as the best ascites batches.

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