Immunofluorescence labeling of LG:

LGs from male NOD and BALB/c mice at the indicated ages were retrieved following euthanasia; fixed in 4% paraformaldehyde for 3 h, transferred to 30% sucrose and incubated overnight at 4^oC. Fixed glands were inserted into OCT compound and flash-frozen on dry ice. The blocks were cryosectioned at 5 μm thickness and mounted onto a glass slide. The sections were then permeabilized with 0.1% Triton X-100 for 10 min and 1% SDS for 5 min. Following this, the sections were blocked with 1% BSA for 1 h and later incubated with primary and secondary antibodies for a period of 1 h each at 37^oC. Between each incubation, the slides were washed with PBS three times for 5 min each. Finally, the samples were mounted with ProLong anti-fade and imaged using a Zeiss LSM 800 confocal fluorescence microscope (Zeiss, Thornwood, NY). Average Mean gray values of 3 random acini from 3 randomly selected images obtained from each of 3 mice in each age group was measured using ImageJ software (National Institutes of Health, http://imagej.nih.gov/ij) to quantify the expression level of different proteins tested.