The following protocol was used for immunostaining tissue sections with anti-Iba1 (Wako, 019–19 471), anti-CD68 (Bio-Rad, MCA1957), anti-GFAP (Millipore, AB5541), anti-NeuN (Cell Signaling, D4640) antibodies. Comparable sections of ATG7KO and littermate control mice were chosen for staining. Selected sections were put on a slide and dried for 5 min. The sections were then washed with 1× PBS three times, 5 min each. The sections were then blocked and permeabilized with blocking buffer (0.3% Triton X-100 and 10% BSA in 1× PBS) for at least 1 h at room temperature. Then antibodies were diluted in blocking buffer and the sections were incubated with diluted antibodies overnight at 4°C. The following morning, the sections were washed with 1× PBS three times, 5 min per wash. Fluorophore-conjugated secondary antibodies were diluted in blocking buffer. The sections were incubated with secondary antibody for 2 h at room temperature and then washed with 1× PBS twice, 5 min per wash, then counterstained with DAPI (Sigma-Aldrich, D9542-1MG) diluted in 1× PBS for 5 min at room temperature, and then washed three times, 5 min per wash. Fluoromount G (SouthernBiotech, 0100-01) was overlaid on top of the brain sections and the slides were sealed with nail-polish. A confocal microscope (Zeiss LSM 880) was used to acquire images. A 25× water lens was used to collect images. To collect full-scale images, bounding grids were created. The grid was chosen to have the cortical layer directly above the hippocampus as well as the entire hippocampus. The z-layer was also added for image acquisition and a 15-μm-thick z-layer was chosen to image. The z-layer was chosen to have the maximum intensity. Images were taken in intervals of 1-μm thickness. Following acquisition, czi files were processed for airyscan processing, stitching of tiles, and to have maximum intensity projection. This was done by using ZEN Black 2.3. TIF format images were downloaded using ZEN Blue 2.3. For cell counting, ImageJ software was used and the “analyze particles” option was used to count cells. Data are presented to show cells per 105 μm2. The sample size is six (three Males, three Females) for both ATG7KO mice and littermate controls.

Myelination was evaluated with anti-myelin basic protein (MBP; BioLegend, 808401), anti-proteolipid protein (PLP; Abcam, AB28486), and anti-myelin ODC glycoprotein (MOG; Millipore, MAB5680) antibodies, with a slightly modified protocol. Briefly, brain sections were stained in a free-floating condition (to have maximum antibody penetration) in a 96-well plate. The sections were incubated with blocking buffer (5% FBS and 2% Triton X-100 in 1× PBS) for 2 h at room temperature. Then, primary antibodies were diluted in the same blocking buffer and the sections were incubated with diluted antibodies for 2 h at room temperature. Following primary antibody incubation, the brain sections were washed using 1× PBS three times, 5 min per wash. After the PBS washes, secondary antibodies were diluted in the same blocking buffer and incubated for 2 h at room temperature, followed by washing and then counterstaining with DAPI (Sigma-Aldrich, D9542-1MG). Brain sections were mounted with Fluoromount G (SouthernBiotech, 0100-01). An epifluorescence microscope (Zeiss Imager M2; Neurolucida 2018 by MBF Bioscience) was used to acquire images. A 20× air lens was used to collect images. To collect full-scale images, a contour containing the entire brain section was drawn, and the brain sections were focused at multiple locations to have the entire section focused. For imaging intensity analysis, the “measure” option of ImageJ software was used to quantify staining intensity. Data are presented as percentage of wild-type. Three comparable sections were stained and imaged for each animal, and thus 18 images in total were imaged and quantified for each group.

For staining with anti-Olig2 (Millipore, AB9610) and anti-CC-1 (CalBioChem, OP80-100UG) antibodies, tissue sections were first processed via antigen retrieval. Antigen retrieval was achieved by heating 10 mm sodium citrate buffer (pH 6) in a container to boiling. The container was then removed from the heat source and glass slides containing the tissues were submerged in the warm buffer. The container was then put on a shaker with mild shaking. The slides were incubated for 20–25 min. Postantigen retrieval, tissue slices were washed with 1× PBS three times, 5 min each. Then, tissue slices were incubated with blocking buffer (2% goat serum + 1% Triton X-100 in 1× PBS) for at least 2 h at room temperature. The tissues were incubated with diluted primary antibodies in the same blocking buffer at 4°C overnight. After washing to remove the unbound primary antibody, the sections were incubated with secondary antibody for 2 h at room temperature, followed by washing, counterstaining, and mounting in Fluoromount G (SouthernBiotech, 0100-01). A confocal microscope (Zeiss LSM 880) and 25× water lens was used to collect images. To collect full-scale images, bounding grids were created. The grid was chosen to have the cortical layer directly above the corpus callosum and striatum. The z-layer was also added for image acquisition and a 15-μm thick z-layer was chosen to image. The z-layer was chosen to have the maximum intensity. Images were taken at intervals of 1-μm thickness. Following acquisition, czi files were processed for airyscan processing, stitching of tiles, and to have maximum intensity projection. In total, 18 images were acquired and quantified for each group. For cell counting of Olig2+/CC1+ cells, ImageJ software was used and the “analyze particles” option was used to count cells. Data are presented to show cells per 0.5 mm2.

The nodes of Ranvier (red) were positively stained for sodium channel using anti-NaV1.6, whereas the paranodes (green) that flank the nodes of Ranvier were labeled with anti-CASPR antibody. For tissue section staining with anti-NaV1.6 (Alomone labs, ASC-009) and anti-CASPR (Neuromab, Clone K65/35, 75-001) antibodies, the tissue sections were first put through antigen retrieval as described above, followed by blocking and permeabilization with blocking buffer (5% goat serum and 0.5% Triton X-100 in 1× PBS) for at least 2 h at room temperature. Tissues were then incubated with primary antibodies diluted in the same blocking buffer for 48 h at 4°C, followed by incubation with the secondary antibodies. Confocal images were acquired with a 63× oil lens. Z-stack images of 4 μm thick were taken at 0.20-μm intervals. Images were processed by using ZEN Black 2.3. For each animal, one section was stained and three different single images were taken for the cortical and corpus callosum regions. The length of the nodes of Ranvier was quantified using Imaris software. Maximum Intensity-projected czi images that were acquired from confocal microscopy were uploaded to the Imaris software. The length of the nodes was calculated automatically. The list of all sizes of node length calculated from Imaris was downloaded and used to calculate the average length and length distribution.

Note: The content above has been extracted from a research article, so it may not display correctly.



Q&A
Please log in to submit your questions online.
Your question will be posted on the Bio-101 website. We will send your questions to the authors of this protocol and Bio-protocol community members who are experienced with this method. you will be informed using the email address associated with your Bio-protocol account.



We use cookies on this site to enhance your user experience. By using our website, you are agreeing to allow the storage of cookies on your computer.