Rabbit anti prox1

Immunofluorescence staining was performed as described previously [24 (link)]. Briefly, 50-μm-thick vibratome or 4-μm-thick wax-embedded sections or MDCK cells were blocked and incubated with one or more primary antibodies. The following primary antibodies were used: rabbit anti-Prox1 (Millipore; Billerica, MA, USA), rabbit anti-LYVE-1 (Research Diagnostics; Flanders, NJ, USA), rabbit anti-NKCC2 (courtesy of Dr. Mark A. Knepper, NIH), goat anti-aldose reductase (AR; courtesy of Dr. Peter Kador, NIH), rabbit anti-AQP2 (Millipore), rabbit anti-CLC-K (Millipore), rabbit anti-AQP1 (Millipore), rat anti-CD31 (BD Bioscience; Franklin Lakes, NJ, USA), goat anti-THP (Mpbiomedicals; Aurora, OH, USA), mouse anti-PCNA (Dako; Carpinteria, CA, USA), and rabbit anti-TonEBP. Appropriate Alexa 488- (Invitrogen; Carlsbad, CA, USA), Cy3-, or Cy5- (Jackson ImmunoResearch Laboratories; West Grove, PA, USA) conjugated secondary antibodies were used for fluorescent detection; nuclei were stained with 4',6-diamidino-2-phenylindole (DAPI; Roche Molecular Biochemicals; Indianapolis, IN, USA). For wax-embedded sections, citrate buffer (pH 6.0) was used for antigen retrieval.

Explant cultures were harvested and fixed with 4% paraformaldehyde for 30 min and then treated with 0.1% Triton X-100 plus 10% donkey serum for 1 h. The explants were then incubated with the following primary antibodies for 24 h at 4°C: rabbit anti-myosin7A (1:100; Proteus Biosciences, Ramona, CA, USA), mouse anti-myosin7A (1:200; Developmental Studies Hybridoma Bank, Iowa City, IA, USA), rabbit anti-Prox1 (1:1,000; Millipore), mouse anti E-cadherin (against the C-terminus, 1:200, BD Transduction Laboratories, San Jose, CA, USA), and goat anti-p120-catenin (1:200; Santa Cruz Biotechnology, Dallas, TX, USA). The explants were washed three to five times in PBS and incubated with secondary antibodies overnight at 4°C in the dark. The secondary antibodies included donkey anti-mouse/rabbit/goat Alexa Fluor 555 (1:1,000), donkey anti-mouse/rabbit Alexa Fluor 488 (1:1,000) and/or donkey anti-mouse/rabbit (H+L) Alexa Fluor 647 (1:1,000; Jackson ImmunoResearch, West Grove, PA, USA). EdU staining was performed as described previously (Zhang et al., 2012 (link)), and immunofluorescence staining was performed immediately following EdU staining.

Briefly, primary neurons were fixed with 4% PFA at DIV21 for 15 min at room temperature, and blocked in PBS with 1% horse serum and 0.1% Triton X-100 (PHT) for 1 hr. Cells were incubated with primary antibodies in PHT overnight at 4°C, followed by 1-hr incubation with fluorophore-conjugated secondary antibodies at room temperature. Primary antibodies used included: rabbit anti-Prox1 (1:5000, Millipore), mouse anti-myc (1:1000, Developmental Studies Hybridoma Bank at the University of Iowa), chicken anti-GFP (1:1000; AVES, Tigard, OR), goat anti-GFP (1:500; Rockland, Limerick, PA), guinea pig anti-vGlut1 (1:1000; Millipore), mouse anti-PSD95 (1:500; Millipore), rabbit anti-VGAT (1:1000; Synaptic Systems), and mouse anti-gephyrin (1:1000; Synaptic Systems). Species-specific and Alexa Fluor-conjugated secondary antibodies (Life Technologies) were used at 1:1000.

Histological and immunofluorescence staining on cryosections was performed as described previously (de Melo et al., 2005 (link)). Primary antibodies used were: mouse anti-BrdU (1:200, Chemicon), mouse anti-BRN3A (1:200, Santa Cruz), goat anti-BRN3B (1:200, Santa Cruz), rabbit anti-caspase 3 (1:500, Cell Signaling Technologies), rabbit anti-DLX2 (1:400, C199 affinity purified), mouse anti-ISLET1 (1:600, DSHB, University of Iowa), rabbit anti-phosphohistone H3 (1:1000, Upstate), rabbit anti-PROX1 (1:500, Chemicon), rabbit anti-PAX6 (1:800, Covance) and mouse anti-syntaxin (1:6000, Sigma). Secondary antibodies and fluorescent tertiary molecules used were: FITC-conjugated goat anti-rabbit (1:200), biotin-SP-conjugated goat anti-rabbit (1:200), biotin-SP-conjugated goat anti-mouse (1:200) (Jackson ImmunoResearch), streptavidin-conjugated Oregon Green 488 (1:200) and streptavidin-conjugated Texas Red (1:200) (Molecular Probes). Negative controls omitted the primary antibody. TUNEL staining used the In Situ Cell Death Detection Kit, TMR Red (Roche Diagnostics). Non-radioactive digoxigenin in situ RNA hybridization was performed as described previously (de Melo et al., 2005 (link)).