Fluorescent mounting media

MDA‐MB‐231 cells expressing STXBP6 and its corresponding control cells were grown on culture slides (BD Falcon) to identify STXBP6‐mediated autophagy induction. At 48 h post treatment with 20 nm everolimus, cells were fixed with 4% paraformaldehyde for 15 min at room temperature and permeabilized in ice‐cold methanol for 15 minutes. Cells were then blocked in 5% normal horse serum for 1 h at room temperature and probed with rabbit anti‐LC3 and mouse anti‐STXBP6 antibodies overnight at 4°C. Cells were incubated with anti‐mouse IgG‐AlexaFluor‐488 (green) and anti‐rabbit IgG‐AlexaFluor‐594 (red) (1:200) secondary antibodies for 1 h at room temperature and then loaded with DAPI for nuclear staining. The coverslips with the attached cells were finally mounted on glass slides with fluorescent mounting media (Vector Laboratories). The fluorescence images were captured using a LSM710 confocal laser scanning microscope (Carl Zeiss).

Whole kidneys were collected at P21 and frozen in OCT. Tissue was cut in 20 μm sections using a cryostat and directly mounted onto slides. Sections were fixed with ice-cold acetone/methanol (50:50 mixture) prior to incubating in blocking buffer (5% BSA in 1xPBS with 0.1% Triton X-100) for 1 h at room temperature. The sections were then incubated overnight at 4°C with FITC-conjugated tomato-lectin (1:200; Vector Laboratories, Burlingame, CA, United States). Slides were then washed and coverslipped with fluorescent mounting media (Vector Laboratories, Burlingame, CA, United States) and images were captured using Eclipse Ti microscope/DS-Ri1 CCD color camera and NIS analysis software (Nikon Instruments). Five images per animal were then analyzed for the number of stain-specific positive pixels using Adobe Photoshop (Adobe Inc.). Briefly, images were converted to grayscale, adjusted to a set threshold equal to the antibody staining and the number of pixels calculated. The data were averaged per group and are presented a number of positive pixels.

For dual immunostaining, sections were labeled with rabbit polyclonal anti-MeCP2 (1:100) or anti-MeCP2 pS421 (1:100) with goat polyclonal antiglial fibrillary acidic protein (anti-GFAP) (1:200; Cat no. ab53554; RRID: AB_880202, Abcam), mouse monoclonal anti-NeuN (1:500; Millipore, Cat. no. MAB377; RRID: AB_229877), mouse monoclonal anti-Nestin (1:500; Cat no. 556309; RRID: AB_396354, BD Biosciences), or goat polyclonal anti-DCX (1:100; Cat no. sc-8066; RRID: AB_2088, Santa Cruz). Sections were then labeled with antigoat IgG-rhodamine (1:40; Santa Cruz) or antimouse IgG–fluorescein isothiocyanate (FITC) (1:40; Santa Cruz) for 1 h at 37°C and mounted using fluorescent mounting media (Vector Laboratories).
The fluorescent double staining was performed by MeCP2, MeCP2 pS421, or AD7c-NTP with DAPI to explore their intracellular distribution. The sections were first incubated with the primary antibody against MeCP2, MeCP2 pS421, or AD7c-NTP and then with the secondary antibody as described above. After further stained with DAPI (1 μg/ml) for 10 min at room temperature, the sections were finally washed, mounted, and coverslipped.
All the sections were detected on a confocal microscope (TCS SP5, Leica, Heidelberg) at excitation of 650 nm and an emission of 670 nm (Cy5), 535 and 565 nm (rhodamine), 490 and 525 nm (FITC), and 358 and 461 nm (DAPI).