16
remixed cultures along with straight purified and control
unpurified cultures were immunostained live with A2B5 and a
rhodamine-goat anti-mouse secondary antibody as above.
Scored were the percent of the DiO(+) cells that were also
A2B5(+) in remixed cultures and percent A2B5(+) in purified
and unpurified cultures.
Immunohistochemistrv
Immunostaining with monoclonal antibody A2B5 was
performed by incubating coverslips in 1/25 dilution of
hybridoma supernatant in Tyrode's + 10% HI-FBS on ice for
30 min. Coverslips were then rinsed with Tyrode's 3x, fixed
with 1% formaldehyde in phosphate-buffered saline (PBS) for
30 min., and rinsed 3x in PBS. Either fluorescein-goat
anti-mouse IgM or rhodamine-goat anti-mouse IgM diluted
1/50 + 5% NGS in PBS for 30 min. was used as the secondary
antibody. This procedure results in specific labelling of
neurons in monolayer cultures without labelling the
flattened glial cells.
Immunostaining of cells and monolayers with anti-
galactocerebroside (GC) was performed essentially the same
as with A2B5 above. A dilution of 1/50 of a purified
monoclonal antibody against galactocerebroside (Ranscht et
al., 1982; kindly provided by Dr. Steve Pfeiffer, Univ. of
Conn. Health Center) was used. FITC-GAM IgG + IgM
(Boehringer) was used as the secondary antibody.