Picrosirius red stain and collagen quantification

Frozen sections placed onto Fisherbrand Superfrost charged microscope slides were fixed in 10% neutral buffered formalin for 5 min and then rinsed in distilled water. Slides were then incubated in Solution B (Direct Red 80/2 4 6-Trinitrophenol) from the Picrosirius Red Stain Kit (Polysciences, Inc., Mount Arlington, NJ, Catalog no. 24901) for 15 min. After a thorough rinse in distilled water, the slides were placed in Solution C (0.1 N hydrochloride acid) for 2 min. Slides were counterstained for 5 min with 1% Fast Green in 1% glacial acetic acid from Poly Scientific (Bay Shore, NY, Catalog no. S2114) using a 1:10 dilution in deionized water. Finally, the slides were rinsed again in distilled water, dehydrated in graded ethanol, cleared in xylene, and mounted with coverslips using Cytoseal 60 media from Thermo Scientific (Catalog no. 8310). Images were taken using the AxioVision 4.9.1 software. Representative images of DIA tissue sections in mdx mice treated with rAAVrh74.MHCK7.micro-dystrophin were compared to mdx-LR mice at 20X magnification.

For analysis of Sirius red staining and percent collagen quantification, the contrast between the red and the green colors was enhanced using Adobe Photoshop. The color deconvolution plugin in the ImageJ software program was selected, and the RGB color deconvolution option was used. The red image includes all connective tissue from the Sirius red stain. The green image includes all muscle from the Fast Green counterstain. Only the red image and the original image were used. A threshold was applied to the images to obtain black and white images with areas positive for collagen in black and negative areas in white. Using the measure function, the area of collagen was calculated. The total tissue area was determined by converting the original image to “8-bit” and adjusting the threshold to 254 (one unit below completely saturating the image). The total tissue area was measured as done previously, and total area was recorded. Quantification of collagen accumulation in the DIA of WT mice, mdx-LR, and mice treated with low (2 × 10¹² vg), intermediate (6 × 10¹² vg), and high (1.2 × 10¹³ vg) doses was calculated by dividing the area of collagen by total tissue area and used to determine the mean percentage for each individual (n = 5, low dose; n = 8, intermediate and high dose; n = 6, mdx-LR; and n = 5, WT).