Computational protocol: Impaired myelination and reduced brain ferric iron in the mouse model of mucolipidosis IV

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[…] To obtain brain tissue for histological examination, 10-day-old, 2- and 7-month-old Mcoln1−/− and control mice were transcardially perfused under isoflurane anesthesia with ice-cold PBS followed by 4% paraformaldehyde in PBS. Brains were postfixed in 4% paraformaldehyde in PBS for 24 h, washed with PBS, cryoprotected in 30% sucrose in PBS overnight, frozen in isopentane and stored at −80°C. Brains were bisected along the midline, and one hemisphere was examined histologically. Coronal sections 40 μm thick were cut using a Microm freezing microtome and collected into 96-well plates containing TBSAF (TBS, 30% ethylene glycol, 15% sucrose and 0.05% sodium azide). These sections were stored at 4°C prior to any staining procedures. For experiments, the corresponding serial sections were used for each brain. For PLP, MBP and SMI312 staining, the sections were microwaved in citrate buffer (10 mM citric acid and 0.05% Tween 20, pH 6.0) at low power for 10 min for antigen retrieval, blocked in 0.5% Triton X-100 and 5% normal goat serum (NGS) in PBS and incubated with primary antibodies diluted in 1% NGS overnight. The following primary antibodies were used: PLP rabbit polyclonal, 1:500, ab28486; MBP (SMI99), mouse monoclonal, 1:1000, ab24568; SMI312 (pan axonal neurofilament) mouse monoclonal 1:1000, ab24574; all from Abcam (Cambridge, MA, USA). Sections were incubated with secondary antibodies in 1% NGS for 1.5 h at room temperature. The following secondary antibodies were used: goat anti-mouse AlexaFluor 488 and donkey anti-rabbit AlexaFluor 555 (1:500; Invitrogen, Eugene, OR, USA). For APC-CC1 staining, no antigen retrieval was performed. After blocking, the sections were incubated with anti-APC-CC1 antibody (mouse monoclonal, 1:100, OP80; Calbiochem, Billerica, MA, USA), followed by goat anti-mouse AlexaFluor 633 (1:500; Invitrogen, Eugene, OR, USA). Sections were counterstained with NucBlue nuclear stain (Life Technologies, Eugene, OR, USA) and mounted on microscopic slides.For PLP, MBP and SMI312 staining, images for analysis were acquired on a Nikon 80i upright epifluorescence microscope with Hamamatsu Orca CCD camera (Nikon, Tokyo, Japan) with 5× objective and NIS-Elements 4.2 software (Nikon, Tokyo, Japan) using an automated stitching function. The exposure time was set to avoid saturation and was the same for all sections (wild-type and Mcoln1−/− sections within the same immunohistochemistry experiment). Image analysis was performed using FIJI software (NIH, Bethesda, MD, USA). Areas of interest (whole hemi-section, cortical region or corpus callosum) were selected in each section. Area and mean pixel intensity values were compared between genotypes using GraphPad Prizm 5 software (GraphPad, La Jolla, CA, USA) using Student's t-test.Quantitative morphological analysis of corpus callosum was done using the AnalyzeSkeleton plugin of ImageJ (). Regions of interests (ROI; 200 pixels ×200 pixels) of corpus callosum were selected on images of PLP-stained sections of 10-day-old brains. Selections were converted to 8-bit images, made binary and skeletonized using the Skeletonize 2D/3D option and analyzed using the AnalyzeSkeleton plugin. An investigator was blinded to genotypes. Total counts of number of branches, junctions, end-point voxels, junction voxels, slab voxels, triple and quadruple points, average and maximal branch lengths per ROI per mouse were averaged across animals in genotype groups (n=5 WT and n=4 Mcoln1−/−) and compared in GraphPad Prizm 5 software using Student's t-test.For APC-CC1 analysis, we acquired images of two non-overlapping fields of view in somatosensory or motor cortex and corpus callosum using a 20× objective (HCX PL APO CS 20.0×0.70 DRY UV) and confocal laser scanning microscope Leica TCS SP5 (Leica Microsystems Inc., Wetzlar, Germany). The number of CC1+ cells was counted in FIJI in corresponding areas of interest (cortex or corpus callosum). CC1 cell density was expressed as the number of cells per square millimeter of the tissue. Statistical analysis of data (two-way ANOVA with Bonferroni correction to assess the effect of genotype and age) was performed using GraphPad Prizm 5. […]

Pipeline specifications

Software tools AnalyzeSkeleton, ImageJ
Applications Laser scanning microscopy, Microscopic phenotype analysis
Organisms Mus musculus
Diseases Mucolipidoses, Lysosomal Storage Diseases, Neurodegenerative Diseases, Leukoencephalopathies
Chemicals Iron