110
THOMAS LIEHR
Outline
1. Freshly extracted hairs are incubated in acetic acid and centrifuged to
remove the hair root cells from the hairs themselves; then the hair root
cells are incubated in fIxative (= methanol/acetic acid) and spotted
onto a dry clean slide.
2. For a FISH approach, these slides are treated with RNase and pepsin
prior to denaturation.
3. Denaturation of the slides is performed for about 5 min at 75°C. Biotinylated and digoxigenated probes are denatured for 5 min and hybridized on the slides for 3 nights at 37°C in a humid chamber. After a
postwashing series, detection of the biotinylated probe is performed
with an FITC-avidin system which leads to a green signal and of
the digoxigenated probe with anti-digoxigenin-rhodamine leading
to red signals. After counterstaining of the nuclei with DAPI and addition of antifade solution, slides can be evaluated under a fluorescence microscope.
Procedure
Hair root cell preparation
1. Freshly extracted hairs can be used at once for the hair root cell preparation or they can be transferred into cell culture medium and stored
there for up to 48 h.
2. Dip the tip of 3 to 6 hair roots into 60 ~l of 50% acetic acid in a 1.5-ml
tube; cut the end of the hair short enough that the tube can be closed
and incubate for 10 min at room temperature (RT).
3. Centrifuge for 5 min at 4000 rpm to remove the hair root cells from the
hairs themselves.
4. Remove the hairs from the tube, add 30 ~l of methanol to the suspension and incubate for 30 min at RT.
5. Pipette the resulting 45 ~l onto two clean dry slides and allow to air dry.
THOMAS LIEHR
Outline
1. Freshly extracted hairs are incubated in acetic acid and centrifuged to
remove the hair root cells from the hairs themselves; then the hair root
cells are incubated in fIxative (= methanol/acetic acid) and spotted
onto a dry clean slide.
2. For a FISH approach, these slides are treated with RNase and pepsin
prior to denaturation.
3. Denaturation of the slides is performed for about 5 min at 75°C. Biotinylated and digoxigenated probes are denatured for 5 min and hybridized on the slides for 3 nights at 37°C in a humid chamber. After a
postwashing series, detection of the biotinylated probe is performed
with an FITC-avidin system which leads to a green signal and of
the digoxigenated probe with anti-digoxigenin-rhodamine leading
to red signals. After counterstaining of the nuclei with DAPI and addition of antifade solution, slides can be evaluated under a fluorescence microscope.
Procedure
Hair root cell preparation
1. Freshly extracted hairs can be used at once for the hair root cell preparation or they can be transferred into cell culture medium and stored
there for up to 48 h.
2. Dip the tip of 3 to 6 hair roots into 60 ~l of 50% acetic acid in a 1.5-ml
tube; cut the end of the hair short enough that the tube can be closed
and incubate for 10 min at room temperature (RT).
3. Centrifuge for 5 min at 4000 rpm to remove the hair root cells from the
hairs themselves.
4. Remove the hairs from the tube, add 30 ~l of methanol to the suspension and incubate for 30 min at RT.
5. Pipette the resulting 45 ~l onto two clean dry slides and allow to air dry.