Isolation of placentae
Timed homozygous matings were set up to produce wild-type (C57BL6 strain) and α7 integrin null (C57BL6-α7βgal strain) embryos and placentae. Pregnant female mice were euthanized in accordance with a protocol approved by the University of Nevada, Reno Animal Care and Use Committee. ED9.5 and ED13.5 placentae were dissected out of the uterus, rinsed in PBS and frozen in liquid nitrogen cooled isopentane.
ED9.5 placentae were cryoprotected by fixing with 4% paraformaldehyde for 2 hours, then washed with phosphate buffered saline (PBS) and placed in a series of 10%, 15%, and 20% sucrose solutions. Placentae were cut cross-sectionally at 10 microns with a Leica CM1850 series cryostat. Hematoxylin and eosin staining was performed following a previously published protocol [28
Wild-type and α7-/- integrin placentae at ED13.5 were fixed in formalin and embedded in paraffin. Samples were processed and stained with hematoxylin and eosin. Images were obtained using a Nikon SMZ800 light microscope (Nikon, Tokyo, Japan), a Spot Slider RT digital camera (Diagnostic Instruments, Sterling Heights, MI) and Spot software (Diagnostic Instruments, Sterling Heights, MI).
For alkaline phosphatase staining, 20 μm sections were stained using an alkaline phosphatase kit according to the manufacturer’s instructions (Sigma Aldrich, St. Louis, MO). Images were acquired using a Zeiss Axioskop 2 Plus fluorescence microscope, Zeiss Axiocam HRc digital camera and Axiovision 4.1 software. Samples were processed and stained with Mason’s Trichrome by the Department of Pathology at the University of Nevada, Reno.
β -galactosidase staining
α7 integrin null mice were produced by replacing exon 1 of the α7 integrin gene with a LacZ reporter gene. Expression of the α7 integrin promoter was detected by β-galactosidase staining. β-galactosidase staining of wild-type and α7 integrin null placentae was performed following a previously published protocol [25
Western blot analysis
Placentae were powdered in liquid nitrogen. Protein was extracted in 2% Triton X-100, 100mM Tris-HCL, 50mM NaCl, 10mM MgCl2, 10mM CaCl2, 1:200 Protease Inhibitor Cocktail Set III (Calbiochem, EMD Biosciences, San Diego, CA) and 1mM PMSF. Protein samples were quantified by a Bradford assay. To detect the α7 integrin, 20 μg of protein was loaded on a 7.5% polyacrylamide gel (BioRad Laboratories Inc. Hercules, CA) under non-reducing conditions and transferred to nitrocellulose membranes. To detect tropomyosin, 50 μg of protein was loaded on a 12% polyacrylamide gel under reducing conditions (addition of 5% β-mercaptoethanol and boiling the sample for 5 minutes) and transferred to nitrocellulose membranes. Smooth muscle myosin heavy chain, the α3 integrin, the α5 integrin, and the α6 integrin were detected by loading 50 μg of protein on a 7.5% polyacrylamide gel under reducing conditions and transferring to nitrocellulose membranes.
The α7 integrin was identified with a rabbit polyclonal antibody anti-α7B (B2 347) (Dr. Stephen Kaufman, University of Illinois, Urbana, IL) at a dilution of 1:2000. Tropomyosin was detected with a mouse monoclonal anti-tropomyosin antibody (Sigma Aldrich, St. Louis, MO) at a dilution of 1:2000. Smooth muscle myosin heavy chain was identified with an anti-smooth muscle myosin heavy chain rabbit polyclonal antibody (Biomedical Technologies, Stoughton, MA) at a dilution of 1:1000. The α3 integrin was detected with an anti-α3 integrin rabbit polyclonal antibody (Chemicon International, Temecula, CA) at a dilution of 1:1000. The α5 integrin was detected with an anti-α5 integrin rabbit polyclonal antibody (Dr. Maria Valencik, University of Nevada, Reno, NV). The α6 integrin was detected with an anti-α6 integrin rabbit polyclonal antibody (Santa Cruz Biotechnology, Santa Cruz, CA) at a dilution of 1:200. Alexa Fluor 680 goat anti-rabbit IgG (Molecular Probes, Eugene, OR) or Alexa Fluor 800 donkey anti-mouse IgG (Molecular Probes, Eugene, OR) was used to detect primary antibodies. Blots were normalized for protein loading by reprobing with sheep anti-gamma actin (Chemicon International, Temecula, CA) or goat anti-Cox-1 polyclonal antibody (Santa Cruz Biotechnology, Santa Cruz, CA). Bands were detected with an Odyssey Imaging System (LiCor Biosciences, Lincoln, NE) and quantified with Odyssey Imaging software.
Placentae were embedded in Tissue-TEK OCT compound (Sakura Finetek USA Inc., Torrance, CA) and 10 micron sections cut with a Leica CM1850 cryostat. Sections were fixed in either cold methanol or 4% paraformalydehyde for two minutes, washed in PBS and then blocked in PBS containing 5% Bovine Serum Albuminin (BSA) for 20 minutes. The α7 integrin was detected with the CA5.5 rat monoclonal antibody (Sierra Biosource, Morgan Hill, CA) at a 1:500 dilution followed by a 1:1000 dilution of FITC-conjugated anti-rat secondary antibody (Jackson ImmunoResearch Laboratories, West Grove, PA). Fibronectin was detected with an anti-fibronectin rabbit polyclonal antibody (Sigma Aldrich, St. Louis, MO) at a 1:400 dilution followed by a 1:500 dilution of FITC-conjugated anti-rabbit secondary antibody (Jackson ImmunoResearch Laboratories, West Grove, PA). A Cy3-labeled anti-smooth muscle actin monoclonal antibody (Sigma Aldrich, St. Louis, MO) was used at a dilution of 1:500 to identify blood vessels located in the placental labyrinth. Sections were mounted in Vectashield+DAPI (Vector Laboratories, Burlingame, CA). A Zeiss Axioskop 2 Plus fluorescence microscope was used to visualize sections and images were captured with a Zeiss Axiocam HRc digital camera and Axiovision 4.1 software. Nuclei were counted in placentae from ED9.5 wild-type and α7 integrin null embryos over 10 random fields at 1000X magnification.
All averaged data are reported as the mean ± standard deviation unless otherwise stated. Student’s t-tests were performed using SigmaStat 1.0 software (Jandel Corporation, San Rafael, CA). A p-value of P<0.05 was considered statistically significant.