Axiozoom v16 stereozoom microscope

In situ hybridization was completed utilizing our previously published procedure.^{8 (link)} Briefly, tissue samples were collected and fixed overnight in 4% paraformaldehyde (PFA), dehydrated in methanol, and stored at −20°C. Tissue was rehydrated, bleached with 6% hydrogen peroxide, incubated in proteinase K (10 μg/ml), fixed in 4% PFA, pre-hybridized in hybridization buffer at 70°C, and then incubated overnight in a RNA probe that recognized the rtTA sequence. Tissue was transferred to a BioLane HTI machine, which performed formamide washes, antibody incubation, and MBST (100 mM maleic acid, 150 mM NaCl, 0.1% Tween-20) washes. Samples were incubated with BM purple for up to 48hr to reveal the in situ hybridization of the RNA probe, fixed in 4% PFA, and stored in 80% glycerol. Tissue was imaged in an AxioZoom.V16 stereozoom microscope (Zeiss).

Embryonic tissue was dissected and briefly fixed in 4% PFA. Whole-mount samples were permeabilized in 0.02% NP-40 and incubated at 37°C for several hours in LacZ stain solution (5mM K₃Fe(CN)₆, 5mM K₄Fe(CN)₆, 2mM MgCl₂, 0.01% Na deoxycholate, 0.02% NP-40, 1mg/ml X-gal). Tissue was post-fixed in 4% PFA overnight at 4°C and stored in 80% glycerol/PBS. Images were taken on AxioZoom.V16 stereozoom microscope (Zeiss).
Tissues for sectioning were dissected, fixed briefly with 4% PFA, incubated in 30% sucrose overnight, and embedded in OCT. Cryoblocks were sectioned at 12μm (Zeiss Microm HM550 cryostat). Tissue was washed with 0.02% NP-40 to permeabilize, and then incubated in LacZ stain solution overnight. Fast red was used to counterstain the tissue. Slides were rinsed in water, dehydrated with ethanol, and washed in xylene. Tissue was mounted with Permount and coverslipped. Images were taken on the AxioScan.Z1 (Zeiss)

Embryonic lungs and kidneys were harvested and subjected to a short fixation in 4% PFA. For whole-mount staining, tissue was incubated in blocking solution (10% sheep serum, 0.1% Triton, PBS) for 1hr and incubated in primary antibodies at 4°C for 24–48hr. Antibodies used were cytokeratin (1:500; Sigma C2562, Novus Biologicals NB120–11213), Six2 (1:500; Proteintech 11562–1-AP), and vimentin (1:500; Abcam ab92547). Samples were washed in PBST (0.1% Triton, PBS) to remove and residual primary antibody for several hours, and then incubated in the corresponding secondary antibody for 24–48hr at 4°C. Tissue was again washed in PBST for several hours to remove any remaining secondary antibody, and then imaged on an AxioZoom.V16 stereozoom microscope (Zeiss).
For cryosections with native fluorescence in the tissue, embryonic kidneys were harvested, fixed in 4% PFA, incubated overnight in 30% sucrose, embedded in OCT, and stored in −80°C. Cryoblocks were sectioned at 12μm thick (Zeiss Microm HM550 cryostat), placed on glass slides (VWR Superfrost Plus Micro Slide), and stored at −80°C. To remove the OCT, slides were washed in PBS, and then incubated in Hoechst 33342 to stain nuclei. Tissue sections were imaged on a confocal SP8 microscope (Leica) to capture native fluorescent signal.