Vectasheild

Fixed hemibrains were cryoprotected in 30 % sucrose, frozen, and sectioned in the horizontal plane at 20 μM using a cryostat. Sections were soaked in PBS for 5 min, then immunolabeled with antibodies directed against TSPO (rabbit monoclonal, 1:1000; Abcam), glial fibrillary acidic protein (GFAP; rat monoclonal, 1:1000 dilution, 2.2B10, Zymed), or anti-ionized calcium-binding adaptor molecule-1 (IBA-1; rabbit polyclonal, 1:1000 dilution, WAKO). Non-specific staining was blocked in blocking reagent (PerkinElmer) for 1 h at room temperature, then immunolabeled overnight at 4 °C with primary antibodies. Sections were then washed three times in PBS for 5 min and then incubated with anti-rabbit or anti-rat IgG biotin (1:1000) for 1 h at room temperature. Immunoreactivity was visualized using tetramethylrhodamine-labeled tyramide signal amplification (PerkinElmer). Sections were mounted with VectaSheild (Vector Laboratories), and coverslips were sealed with enamel. Photomicrographs were captured with a fluorescence microscope/digital camera (BZ-X700, Keyence) at ×20 magnification and then thresholded at a predetermined, constant value using NIH Image 1.61 to create a binary image identifying positive and negative immunolabeling and the intensity assessed.

Cells were cultured on sterile, untreated glass coverslips, as above, in the presence or absence of HDL (100 µg protein/ml) for different times. The cells were then washed twice with PBS, fixed with 3% paraformaldehyde pH 7.4 for 30 min and permeabilized with 0.1% Triton-X100 for 5 min. at room temperature. F-actin was stained using 25units/ml Alexafluor488 phalloidinin PBS for 30 min at room temperature. The coverslips were then mounted using Vectasheild (Vector Laboratories Canada Inc. Burlington, ON, Canada) and imaged using a Zeiss Axiovert 200 M fluorescence microscope with standard FITC filters using a 40× objective.