For in situ hybridization (ISH), mice were perfused
with 4% paraformaldehyde in a solution of 0.1% diethyl pyrocarbonate in PBS
(PBS-DEPC), post fixed for 1 h at RT, washed 3 times with PBS-DEPC and
cryopreserved overnight in 30% sucrose in PBS-DEPC. Spinal cords were
embedded in Tissue-Tek and stored at —80°C. Spinal cords were
then cryosectioned at 16 μm, and sections were hybridized with an
antisense RNA probe overnight at 64°C. Sections were washed twice in
a solution of 1 × saline-sodium citrate buffer, 50% formamide, and
0.1% Tween-20 at 64°C for 30 min and blocked with a solution of 0.1%
Tween 20 in maleic acid buffer (MABT) containing 2% blocking reagent and 10%
inactivated sheep serum for 2 h at RT. Sections were then incubated
overnight with sheep α-digoxigenin-alkaline-phosphatase Fab fragments
(1:2000; Roche), washed twice in MABT and revealed with a staining solution
of NBT (1:500, Roche) and BCIP (1:600, Roche). An Olympus VS-120 Virtual
Slde Scanning Microscope was used for imaging. For double staining analyses,
tdTomato fluorescence was imaged before ISH was performed. ISH images were
later pseudo-colored and superposed onto the tdTomato signal in Photoshop
(Adobe Systems). For quantification, three sections from each of three
spinal cords were analyzed per condition, and only cells with clearly
visible nuclei were scored.
with 4% paraformaldehyde in a solution of 0.1% diethyl pyrocarbonate in PBS
(PBS-DEPC), post fixed for 1 h at RT, washed 3 times with PBS-DEPC and
cryopreserved overnight in 30% sucrose in PBS-DEPC. Spinal cords were
embedded in Tissue-Tek and stored at —80°C. Spinal cords were
then cryosectioned at 16 μm, and sections were hybridized with an
antisense RNA probe overnight at 64°C. Sections were washed twice in
a solution of 1 × saline-sodium citrate buffer, 50% formamide, and
0.1% Tween-20 at 64°C for 30 min and blocked with a solution of 0.1%
Tween 20 in maleic acid buffer (MABT) containing 2% blocking reagent and 10%
inactivated sheep serum for 2 h at RT. Sections were then incubated
overnight with sheep α-digoxigenin-alkaline-phosphatase Fab fragments
(1:2000; Roche), washed twice in MABT and revealed with a staining solution
of NBT (1:500, Roche) and BCIP (1:600, Roche). An Olympus VS-120 Virtual
Slde Scanning Microscope was used for imaging. For double staining analyses,
tdTomato fluorescence was imaged before ISH was performed. ISH images were
later pseudo-colored and superposed onto the tdTomato signal in Photoshop
(Adobe Systems). For quantification, three sections from each of three
spinal cords were analyzed per condition, and only cells with clearly
visible nuclei were scored.