Computational protocol: AGPAT2 is essential for postnatal development and maintenance of white and brown adipose tissue

Similar protocols

Protocol publication

[…] E18.5 embryos and newborn mice were euthanized by CO2 anesthesia following cervical decapitation. Immediately, they were rinsed with 1X PBS and fixed overnight in 4% PFA/PBS and then transferred into 30–18% sucrose/PBS gradient. For histological studies in dorsal skin and interscapular BAT (iBAT) of P0-P6.5 mice, tissues were fixed in 4% PFA/PBS and then embedded in paraffin. Cryo and paraffin embedding, sectioning, H&E and Oil Red O staining were performed at UTSW Molecular Pathology Core. For Perilipin-1 and MAC-2 immunofluorescence in AT, sections were deparaffinized in xylene and rehydrated in a graded series of ethanol followed by dH2O. Antigen unmasking was carried out by heating slices in 10 mM sodium citrate buffer (pH 6.0) at 95–99 °C for 10 min. Tissue sections were blocked and then incubated overnight at 4 °C with primary antibodies. After the washing steps, fluorochrome-conjugated secondary antibodies were incubated for 1 h at room temperature. For immunofluorescence detection in cultured cells, MEFs were seeded on glass coverslips and adipogenic differentiation was induced as described above. At the indicated days, differentiated MEFs were fixed in 4% PFA, washed with PBS and permeabilizated/blocked in 0.3% Triton X-100; 3% BSA/PBS. Primary and secondary antibody incubation steps were performed as described above. Finally, all stained slides and coverslips were mounted with ProLong® Gold Antifade Reagent with 4′,6′-diamidino-2-phenylindole (DAPI) (Molecular Probes). Images were captured with Leica SP5 Tandem Scanner Spectral 2-photon confocal microscope (Leica Microsystems, Inc., Buffalo Grove, IL) and processed with ImageJ (NIH, Bethesda, MD, US) and Bitplane Imaris software v. 7.3.1 (Andor Technology PLC, Belfast, N. Ireland). The following antibodies and dilutions were used: rabbit anti-Perilipin-1 (1:300, Cell Signalling), rabbit anti-Caveolin-1 (1:100, Cell Signalling), rabbit anti-PPARγ (1:100, Cell Signalling) and rat anti-MAC-2 (1:200, Cedarlane). Alexa Fluor® 488 or 594 goat anti-rabbit IgG (H + L) and Alexa Fluor® 488 goat anti-rat IgG (Molecular Probes) were diluted 1:300 in blocking buffer. F-actin was stained with rhodamine phalloidin (1:30 in PBS, Molecular Probes). For neutral lipid staining in MEFs, samples were incubated with 1 μg/ml BODIPY 493/503 (Molecular Probes). [...] To quantify the number and size of adipocytes in histological sections of adipose tissue, adipocyte area was measured on hematoxylin and eosin stained slides using Adiposoft and ImageJ software as previously described . To determine the relative percentage of lipid-laden cells (cells containing LDs) after 6 days of differentiation, MEFs were grown on glass coverslips and differentiated. At day 6, cells were fixed and stained with BODIPY 493/503 to detect neutral lipids, rhodamine-phalloidin to detect cortical actin lining the inner surface of the plasma membrane in adipocytes , ), and DAPI to stain cell nuclei. Samples were imaged using a confocal microscope. For cell counting, five non-overlapping fields were analyzed and quantified using Image J software (each field contained approximately 150 cells). The relative percentage of lipid-laden cells was determined as the ratio of BODIPY/cortical F-actin labeled cells to the total cell number per field. For time-course experiments, lipid-laden cells were monitored under an inverted microscope with phase contrast objectives at different days of differentiation. Images from the sample fields were captured at each time and then analyzed with Image J software. Quantitative image analysis of fluorescence intensity was performed with Image J software as previously described . […]

Pipeline specifications

Software tools ImageJ, Imaris, Adiposoft
Applications Laser scanning microscopy, Microscopic phenotype analysis
Organisms Mus musculus
Diseases Lipodystrophy, Multiple Sclerosis, Cardiovascular Abnormalities
Chemicals Phosphatidic Acids