Axio observer fluorescence microscope

Cells were fixed with 4% PFA in PBS, permeabilised with ice-cold acetone and subsequently blocked in 1.1% BSA in PBS overnight. Cells were subsequently stained using anti-HA (C29F4, Cell Signalling Technology, USA) and anti-PLN (2D12, Thermo Fisher, USA) antibodies at a 1:400 dilution in blocking buffer, overnight. Cells were washed with blocking buffer and incubated with fluorescently labelled secondary antibodies and nuclei were stained with Gold Anti-fade Reagent with DAPI (Invitrogen, USA). Imaging was performed using a Zeiss axio observer fluorescence microscope (Zeiss, Germany). The use of the anti-PLN antibody 2D12 for immunofluorescence studies was validated using adult murine heart tissue (Supplementary Fig. 6).

Cells were fixed with 4% paraformaldehyde (PFA) in Phosphate Buffered Saline (PBS) for 20 min, washed in PBS, permeabilized and blocked using 1% Bovine Serum Albumin (BSA) and 0.1% Triton in PBS. Cells were then incubated with primary antibody for βIII-tubulin (Sigma-Aldrich, Merck, St. Louis, MO, USA) (1:200) for 1 h at room temperature (RT). They were successively washed with 1% BSA in PBS, incubated with Alexa Fluor^® 488 secondary antibody (Invitrogen, Thermo Fisher, Waltham, NA, USA) (1:200) for 1 h at RT, and washed again with 0,1% BSA in PBS. Finally, cells were incubated for 5 min with Hoechst (Invitrogen, Thermo Fisher, Waltham, MA, USA) (1:1000) for nuclei staining. Images were acquired with Zeiss Axio Observer fluorescence microscope (Zeiss, Oberkochen, Baden-Württemberg, Germany).
Morphometric analysis of SH-SY5Y cells was performed on βIII-tubulin immunofluorescence images through ImageJ software (National Institutes of Health, Bethesda, MD, USA), version 1.53t. The NeuronJ plugin was applied for semi-automated neurite tracing. Neurite count and total length values (µm) were expressed as a ratio of the number of cells per image, which was assessed by the Cell Counter plugin.

The Oil Red O (ORO) stock solution was freshly prepared by dissolving 300 mg ORO powder into 100 mL isopropanol. The ORO working solution was then made by diluting ORO stock solution by 60% with deionized water. Immediately before staining, cells were fixed with 10% formalin and incubated with 60% isopropanol in deionized water for 5 min. Then, the ORO working solution was applied to cells for 1 min and washed in deionized water. Cell images were taken by ZEISS Axio observer fluorescence microscope (Zeiss, Oberkochen, Germany) under both bright field and rhodamine channels. The amount of LDs was quantitated by measuring the intensity of red fluorescence using ImageJ [36 (link)].

Cells were fixed for 5 min with 4% paraformaldehyde on day 9 and washed extensively with PBS. The cells were then stained with FITC-conjugated phalloidin (Sigma) and 4’, 6-diamidino-2-phenylindole for 40 min at room temperature. Afterwards, the cells were washed three times with PBS and observed with an Axio Observer fluorescence microscope (Carl Zeiss).

Coronal sections at the level of the anterior commissure were used in all quantitations of cell populations and staining intensities in the dorsal cerebral cortex and corpus callosum. Slides were imaged on AxioObserver fluorescence microscope (Zeiss). Images were processed using CS6 Photoshop software (Adobe) for orientation, false colorization, and overlay/colocalization. Enumeration of cells positive for cytoskeletal, cytoplasmic or membrane proteins was performed manually by counting positive cells using the Photoshop CS6 counting tool. Quantitation of CIC nuclear staining intensity and MBP staining density was performed using FIJI software^{47 (link)} as follows. Images were first processed using Gaussian blur, then background subtracted. Default threshold limits were used in the threshold tool. Images were converted to binary and watershed was run to separate clumped cells. Nuclear staining was quantitated by using the analyzing particles option with separate cutoffs set for each antibody used. For quantitation of MBP expression on tissue sections, FIJI was used by drawing regions of interest on the lateral corpus callosum (cingulum), and the mean integrated density in the regions of interest was calculated.

Immunohistology was carried out as previously described [81 (link)]. In Brief, after euthanizing mice, the thymus was excised under aseptic conditions and frozen in optimal cutting temperature media (Tissue-Tek). Five-micron frozen sections were cut using a Microm HM 550 Cryostat (Thermo Scientific), collected on coated slides, fixed in 4% paraformaldehyde, washed with PBS, and blocked with appropriate sera in PBS. After incubating with appropriate antibodies, sections were washed and incubated with fluorescence dye-conjugated second antibodies and 1 μg/ml of DAPI (Sigma). Stained sections were washed and mounted under a coverslip using Fluoro-gel with Tris Buffer (Electron Microscopy Sciences, Hatfield, Pennsylvania) and examined using an Axio Observer fluorescence microscope (Zeiss).