SDFT samples (A, B) were harvested from a recently deceased foal, using a dissection technique that had been developed during previous work on adult SDFT. The samples were fixed in 4% paraformaldehyde in PBS for 48 hours. They were trimmed into pieces approximately 10mm wide x 15mm long and snap frozen in melting iso-pentane. Sections were cut using a cryotome and mounted on positively charged slides (C-H)

The mounted sections were then stained immunohistochemically using the following protocol:

1. Encircle sections with PAP pen
2. Permeablise cells and block non-specific binding sites by applying a solution of 0.1% Triton X-100 and 1% BSA for 10 minutes at room temperature (RT)
3. Dab off excess moisture
4. Apply enough anti-alpha-SMA primary antibody (RTU-SMA, Novocastra) to cover the section
5. Incubate at RT in humid chamber for 24 hours
6. Rinse in PBS 5 times, 5 minutes per rinse
7. Dab off excess moisture
8. Apply biotinylated anti-mouse IgG secondary antibody (Amersham) at a dilution of 1:200. Incubate in humid chamber for 24 hours at RT
9. Dab off excess moisture
10. Apply staining solution - 0.1 mg / mL RNAse-A (R-5503, Sigma), 5 µg / mL propidium iodide (Molecular Probes), 10 µg / mL streptavidin conjugated Alexa Fluor^® 488 (Molecular Probes) in PBS. Incubate for 24 hours at RT in humid chamber.
11. Rinse in PBS 5 times, 5 minutes per rinse
12. Dab off excess moisture
13. Mount with VECTASHIELD (Vector Laboratories) anti-fade fluorescence mountant under a 40mm x 22 mm no. 1 coverslip (Esco)
14. Seal with nail varnish

The slides were then viewed using a Leica TCS 4D confocal scanning head on a Leica DMRBE microscope. Fluorescence excitation was provided by an Omnichrome Kr/Ar mixed gas laser filtered with a 488/568 nm excitation beamsplitter. Dual detection of propidium iodide (Em = 617 nm, orange) and Alexa Fluor 488^® (Em = 519 nm, green) was attained by using an LP 580 second beamsplitter to separate the long emissive wavelengths from the short emissive wavelengths. Long wavelengths were detected by the second photomultiplier. The short wavelengths were further filtered by the aplication of a BP-FITC filter, which allowed only light in the range 515-530 nm to reach the first photomultiplier.

Non-confocal UV epifluorescence was used to make an initial survey of the stained sections. Continuous confocal scanning was used to optimise the laser power, voltage and offset to generate images with bright features of interest and dark background. Line averaging was set to 16. Image stacks were collected through the 16x 0.5 NA, 40x 1.0 NA, 63x 1.4 NA and 100x 1.4 NA lenses. Z-stepping settings approximately 2.3 x the z resolution of the lenses were used to acheive optimal Nyquist sampling of the fluorescence signals. Images were saved as .tiff stacks and visualised later.

Amira (Indeed - Visual Concepts GmbH) was used to generate 3D projections of the two-channel data. Alpha-SMA immunoreactivity was colour-coded green, while DNA reactivity was colour coded red. Projections were rendered in sequence, and the sequences animated using Fast Movie Processor 1.41 (Gromada.com)