Tissue path superfrost plus gold microscope slides

Heads of 3-day-old (D3) wild type (Dspp^+/+), Dspp^+/−1fs and Dspp^{−1fs/−1fs} mice, and mandibles of 14-day-old (D14) Dspp^+/+, Dspp^+/−1fs, Dspp^{−1fs/−1fs}, Dspp^+/P19L, and Dspp^P19L/P19L mice were harvested, fixed in 4% PFA in PBS at 4 °C overnight. The samples were decalcified at 4 °C in 4.13% disodium ethylenediaminetetraacetic acid (EDTA, pH 7.4) with agitation for 4 days (D3 samples) or 12 days (D14 samples). The samples were then dehydrated by an ethanol series, cleared by xylene, embedded in paraffin, and sectioned at 5 μm thickness. Sections were obtained from maxillary 1st molars and mandibular incisors and placed on Fisherbrand Tissue Path Superfrost Plus Gold Microscope Slides (Fisher Scientific) for histological analysis.

GC muscle was frozen immediately after isolation in isopentane cooled with liquid nitrogen. GC muscle was cryosectioned at a thickness of 10 µm and affixed to Tissue Path Superfrost Plus Gold microscope slides (Fisher Scientific). Sections were fixed in 4% formaldehyde in PBS and then permeabilized in 0.15% Triton-X100 (Fisher Scientific) in PBS. Antibodies to GLUT4 (#MA5-17176, 1:500, Thermo Fisher Scientific) and dystrophin (#ab15277, 1:400, Abcam) were applied simultaneously to the sections for 2 h at RT. After incubation with primary antibodies, sections were washed 3 times in PBS for 5 min each time. Secondary antibodies were applied to the sections for 45 min at RT at a dilution of 1:500. For GLUT4 we used goat anti-mouse Alexa Fluor 488 (Thermo Fisher Scientific), and for dystrophin we used goat anti-rabbit Alexa Fluor 555 (Thermo Fisher Scientific) secondary antibodies. Sections were then washed 3 times in PBS for 5 min each time and mounted in ProLong Glass Antifade Mountant (Thermo Fisher Scientific). Images (n > 10 per mouse) were taken on the Zeiss LSM 880 inverted confocal microscope (Carl Zeiss, Inc.). All the images were captured with the same laser intensities. The Pearson’s correlation coefficient for quantitating colocalization of GLUT4 and dystrophin was calculated for each image using Zen Black software (Carl Zeiss, Inc.).