Conditional disruption of Klf5
Derivation and use of Klf5-LoxP
(Wan et al 2008
) and Le-Cre
(Ashery-Padan et al., 2000
) mice has been described previously. Klf5loxP/loxP, Le-Cre
/- mice were mated with Klf5loxP/loxP
mice to obtain equal proportion of Klf5loxP/loxP, Le-Cre
CN) and Klf5loxP/loxP
(control) offspring. Genomic DNA isolated from tail clippings of these mice was assayed for the presence of the Klf5-LoxP
transgenes by PCR using specific primers. Klf5loxP/loxP
PCR was carried out using primers that can distinguish between the WT allele and the floxed allele (primer 1, CCT GCG TGC AAT CCA TCT TGT TCA ATG GC; primer 2, TCA CCC TCT GCA GAT CTT AGG C; and primer 3, GCT TGG CTC AAA ATT CCG TTC C), as before (Wan et al., 2008
). Gestation was determined by identification of a vaginal plug (E0.5). Mice studied here were on a mixed genetic background and maintained in accordance with the guidelines set forth by the Animal Care and Use Committee of the University of Pittsburgh, Pittsburgh and the ARVO statement related to the humane use of animals in experiments.
Eye tissues from carbon dioxide asphyxiated mice were fixed in freshly prepared 4 % paraformaldehyde (Sigma Chemical Company, St. Louis, MO) in phosphate buffered saline (PBS; pH 7.4) for 24 hours at 4°C and embedded in paraffin. To rule out the inadvertent use of sections from the edges of eyeballs, we started collecting serial sections upon entering the angle tissue on one side, ending while exiting on the other side. We then stained the central sections representing the middle of the eye. 8μm-thick sections were stained with hematoxylin and eosin, or periodic acid-Schiff's (PAS) reagent. For oil red-O staining, 8μm-thick cryosections from OCT embedded adult mouse eyelids were air dried for 60 min, fixed in 4 % paraformaldehyde in PBS for 30 minutes and air dried again. Slides were then placed in absolute propylene glycol for 5 min and incubated in pre-warmed 0.5% Oil-red-O stain for 15 min in 60° C oven, differentiated in 85% propylene glycol, rinsed in two changes of distilled water, counterstained with Meyer's hematoxylin and mounted using aqueous mounting medium. Light microscopy was performed with an Olympus BX60 microscope (Olympus America Inc.) equipped with Spot digital camera (Spot diagnostics instruments Inc., Sterling Heights, CA).
In Situ hybridization
hybridization was performed using 12 μm-thick cryosections from fresh frozen eye tissue in OCT. The sections were fixed in 4% paraformaldehyde, treated with proteinase K (0.2 μg/mL) for 5 minutes, and processed for in situ
hybridization as described earlier (Norman et al., 2004
). Riboprobes were synthesized using a digoxygenin (DIG) RNA labeling kit (Sp6/T7; Roche Molecular Biochemicals, Indianapolis, IN) with linearized plasmid cDNA templates for Klf4 and Klf5. Color development reaction was allowed to proceed until purple color was visible, (approximately 30 to 60 minutes) and reactions for both the sense and antisense riboprobes were terminated at the same time.
Isolation of total RNA, RT-PCR and real time quantitative RT-PCR
Klf5 mRNA was quantitated in the developing mouse cornea by real time quantitative RT-PCR (Q-RT-PCR) using a standard curve generated with serial dilutions of linearized plasmid pCMVSport6-Klf5. Q-RT-PCR was performed using cDNA synthesized with 100 ng total RNA isolated from dissected corneas. The reagents, equipment and software for TaqMan gene expression assays were obtained from Applied Biosystems, Foster City, CA. Q-RT-PCR assays with pre-standardized gene-specific probes were performed in ABI StepOne Plus thermocycler using 18S rRNA as endogenous control and the results analyzed using the software provided by the manufacturer (Applied Biosystems).
Immunoblots and immunofluorescence
Equal amounts of total protein extracted by homogenizing dissected corneas in 8.0 M urea, 0.08 % Triton X-100, 0.2 % SDS, 3% β-mercapto ethanol and proteinase inhibitors and quantified by bicinchoninic acid method (Pierce, Rockford, IL) were separated by electrophoresis in SDS-PAGE gels, transferred to PVDF membranes and subjected to immunoblot analysis. Rabbit anti-KLF5 antibody (Abcam, Cambridge, MA) and goat anti-actin antibody that recognizes a broad range of actin isoforms (Santa Cruz Biotechnology, Santa Cruz, CA) were used at 1:500 dilution in PBST. Horseradish peroxidase- coupled goat anti-rabbit IgG (Invitrogen, Carlsbad, CA) or donkey anti-goat IgG (Santa Cruz Biotechnology, Santa Cruz, CA) antibody was used at 1:5000 dilution. Immunoreactive bands were identified by chemiluminescence following incubation with Super Signal West Pico solutions (Pierce, Rockford, IL).
For immunofluorescence, 8 μm-thick sections from OCT or paraffin embedded eye tissues were fixed in freshly prepared buffered 4 % paraformaldehyde for 30 minutes, blocked with 10 % goat serum in PBST for 1 h at room temperature in a humidified chamber, washed twice with PBST for 5 minutes each, incubated with 1:150 dilution of rabbit anti-KLF5 antibody, 1:100 dilution of rabbit anti-laminin-332 antibody (Abcam, Cambridge, MA), 1:500 dilution of rabbit anti-αA-crystallin antibody, 1:500 dilution of rabbit anti-αB-crystallin antibody, or 1:25 dilution of rabbit anti-Ki67 antibody (Fisher Scientific, Pittsburgh, PA) for 1 h at room temperature, washed thrice with PBST for 10 minutes each, incubated with second antibody (Alexafluor 555 coupled goat anti-rabbit IgG antibody, Molecular Probes, Carlsbad, CA) at 1:1500 dilution for 1 h at room temperature, washed thrice with PBST for 10 minutes each, mounted with Prolong Gold anti-fade reagent with DAPI (Molecular Probes, Carlsbad, CA) and observed with an Olympus Fluoview 1000 confocal system with an Olympus IX81 microscope.