Two fixation procedures were used. Samples were incubated overnight at
4°C in Carnoy fixative (60% ethanol, 30% chloroform, 10% acetic acid) and
stored in 70% ethanol. Alternatively, samples were fixed overnight at 4°C
in 70% methanol, followed by one day at 4°C in 100% methanol, and then
stored at -20°C in methanol.
N=8 wt and N=8 gal3-/- tracheas were paraffin-embedded
for sectioning. Sections (5μm) were de-waxed in a xylene bath, rehydrated
once in isopropanol, then in decreasing ethanol solutions, and were processed
for either immunohistochemistry or immunostaining.
Mucus was detected by Alcian blue staining, and nuclei were then
detected by Nuclear Fast Red incubation. Images were acquired using an Eclipse
90i Upright microscope and a cool SNAP HQ2 CCD color camera (Nikon, Tokyo).
For immunostaining, de-waxed tissue sections were blocked in 1.5% donkey
serum (Sigma) for 1h. Primary antibody incubations were performed at 4°C
for 12h, and secondary antibody incubations at room temperature for 2h, both in
1.5% donkey serum solution. Hoechst 33342 staining (Life Sciences) was used to
detect nuclei. Tissue sections were mounted in Mowiol488 solution. Confocal
images of fixed cells were acquired on a Leica TCS SP5 microscope using a 63x
and a 100x lens (Leica Microsystems, Wetzlar, Germany).
4°C in Carnoy fixative (60% ethanol, 30% chloroform, 10% acetic acid) and
stored in 70% ethanol. Alternatively, samples were fixed overnight at 4°C
in 70% methanol, followed by one day at 4°C in 100% methanol, and then
stored at -20°C in methanol.
N=8 wt and N=8 gal3-/- tracheas were paraffin-embedded
for sectioning. Sections (5μm) were de-waxed in a xylene bath, rehydrated
once in isopropanol, then in decreasing ethanol solutions, and were processed
for either immunohistochemistry or immunostaining.
Mucus was detected by Alcian blue staining, and nuclei were then
detected by Nuclear Fast Red incubation. Images were acquired using an Eclipse
90i Upright microscope and a cool SNAP HQ2 CCD color camera (Nikon, Tokyo).
For immunostaining, de-waxed tissue sections were blocked in 1.5% donkey
serum (Sigma) for 1h. Primary antibody incubations were performed at 4°C
for 12h, and secondary antibody incubations at room temperature for 2h, both in
1.5% donkey serum solution. Hoechst 33342 staining (Life Sciences) was used to
detect nuclei. Tissue sections were mounted in Mowiol488 solution. Confocal
images of fixed cells were acquired on a Leica TCS SP5 microscope using a 63x
and a 100x lens (Leica Microsystems, Wetzlar, Germany).