Preparation of Chicken Embryos
Fertile White Leghorn chicken eggs were obtained from Case Western Reserve University's Squire Valleevue Farm and incubated in humidified air at 38°C. The embryos of stages 19–35 (Hamburger and Hamilton, 1951) were handled in accordance with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health and the institutional IACUC guidelines.
Stock solutions of 1 mM LTR (Molecular Probes, Eugene, OR) in dimethyl sulfoxide (DMSO) were diluted in Hanks' balanced salt solution (HBSS) or Dulbecco's phosphate buffered saline (PBS) to yield a final concentration of 2.5 μM. The embryos were dissected to expose the heart and incubated in the LTR solution for 15 min at room temperature (22°C) while rocking and washed in PBS before fixation. Preliminary experiments were carried out by using 1, 2.5, or 5 μM LTR for 5-, 10-, or 15-min incubations. Staining was observed with all concentrations with the best resolution and least amount of background interference achieved by using 2.5 μM LTR for 15-min incubations.
The embryos were fixed in neutral, buffered formalin solution (10%, Ted Pella, Inc., Redding, CA) for 1 hr at room temperature while rocking, washed extensively with PBS, and stored at 4°C before observation and further processing.
A subset of the embryos were processed by standard procedures for paraffin embedding and sectioning. Serial sections (7-μm thick) were collected on Superfrost Plus slides (Fisher Scientific, Pittsburgh, PA). For certain procedures, embryos were processed for frozen sectioning. Briefly, they were incubated in sucrose solutions for cryoprotection and frozen in TBS tissue freezing medium (Fisher Scientific). Frozen sections (10–20 μm) were also collected on Superfrost Plus slides.
Paraffin and frozen sections were observed and photographed before further treatment to document the LTR staining pattern because subsequent procedures reduced the LTR signal. To delineate the myocardium, specific sections were stained with MF20, a mouse monoclonal antibody specific to myosin heavy chain, or Sr-1, an antibody to sarcomeric actin (DAKO M0874, Carpinteria, CA). The MF20 developed by Dr. D. Fishman was obtained from the Developmental Studies Hybridoma Bank developed under the auspices of the NICHD and maintained by The University of Iowa, Department of Biological Sciences, Iowa City, IA 52242. The secondary antibody for Sr-1 was Alexa fluor 488–conjugated goat α-mouse IgM (Molecular Probes).
Select LTR-stained and formalin-fixed intact embryos were stained for MF20 (van den Hoff et al., 1999). Embryos with their hearts exposed were incubated at 4°C overnight in 1:4 DMSO:methanol solution, rehydrated in a graded alcohol series of 95%, 70%, and 50% methanol for 1 hr each at room temperature, incubated in PBS with 10% normal goat serum (NGS) for 1 hr to block nonspecific staining, incubated with a 1:200 dilution of MF20 overnight at 4°C, washed three times for 30 min in PBS with 10% NGS, incubated for 3 hr at room temperature with a goat anti-mouse IgG antibody labeled with Alexa Fluor 488 (Molecular Probes), washed with PBS, and observed with a fluorescence stereomicroscope or by confocal laser scanning microscopy.
For keratin staining of intact hearts, embryos were processed by using a modification of a previously described protocol (Vrancken-Peeters et al., 1995). LTR-stained embryos with hearts exposed were fixed 1 hr in 10% formaldehyde solution, blocked in 10% NGS and 0.05% Tween-20 in HBSS for 1 hr, incubated overnight at 4°C in 1:250-fold dilution of polyclonal anti-keratin antibody (DAKO Z0622) in blocking buffer, washed in PBS then 3 hr in 1:200 goat anti-rabbit conjugated to Alexa fluor 488 (Molecular Probes), and observed and photographed under a conventional fluorescence stereomicroscope or by using a laser scanning confocal microscope. For sections, stained embryos were fixed in formaldehyde solution and processed for paraffin embedding and sectioning. Alternate sections were collected on separate slides and prepared for immunostaining or observed after removal of paraffin by fluorescence microscopy without further staining.
In selected paraffin sections, the presence of apoptosis was confirmed by using the TUNEL technique. Deparaffinized sections were treated with 2% hydrogen peroxide to quench endogenous peroxidase and stained with the ApopTag peroxidase kit (Intergen, Purchase, NY) using the TUNEL technique (Gavrieli et al., 1992) to identify concentrations of DNA fragments characteristic of apoptosis. Briefly, sections were incubated with proteinase K (20 μg/ml, for 15 min at 25°C), exposed to the TdT (terminal deoxynucleotidyl transferase) enzyme containing reaction buffer to add dUTP-digoxigenin to the 3′-OH end of DNA fragments and incubated with anti-digoxigenin conjugated to peroxidase.
Sections processed with the TUNEL technique were rinsed with PBS and incubated at room temperature for 1 hr in MF20 or Sr-1. The MF20 was diluted with 10% NGS in PBS to a concentration of 1:500. The sections were washed in PBS and incubated in goat anti-mouse IgG Alexa Fluor, 1:250, for 1 hr at room temperature, rinsed, and cover-slipped in Gelvatol solution (Airvol 523 polyvinyl alcohol, Air Products and Chemicals, Inc., Allentown, PA) or glycerol with 2% n-propyl gallate.
Whole-mount embryos were observed by using a Leica MZFLIII stereomicroscope or a laser scanning confocal microscope (Zeiss LSM 410; Axiovert 100; Zeiss 5× Plan-Neofluor; WD 13.5 mm; excitation lines 488 nm for green and 568 nm for red). For the projected image in Figure 4, an overlay was made of two projected images by using the red and green filters. Each projected image was the combination of 50 slices of 66 microns per slice. The embryo sections were observed by using a Leica DMLB microscope. Digital images were obtained by using a Spot RT digital camera and software v3.1 (Diagnostic Instruments, Inc., Sterling Heights, MI). Images were imported and brightness adjusted and, when necessary, overlaid by using Adobe Photoshop 6.0 or 7.0.1. All observations reported were observed in tissues from at least three different embryos.