Rabbit anti abca1

After animals were deeply anesthetized with isoflurane, they were perfused with 100-300 ml of 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4). The brain was harvested, postfixed at 4°C for 24 h, and cryoprotected in 30% sucrose overnight. The tissues were sectioned at the thickness of 30 mm on a cryostat. After being blocked with PBS containing 5% goat serum and 0.3% Triton X-100 for 1 h at 37°C, the sections were incubated overnight at 4°C with rabbit anti-Apoe(CST, 1:100, catalog number: 49285) or rabbit anti-Abca1(Novus Biologicals, USA 1:200, catalog number: NB48-105) or rabbit anti-Hexb(Thermo Fisher, 1:100, catalog number: PA5-101082). The sections were then incubated with goat anti-rabbit IgG conjugated with Cy2 (Jackson ImmunoResearch, USA, 1:500, catalog number:111-225-144) for 1 h at room temperature. Control experiments included omission of the primary antiserum and substitution of normal rabbit serum for the primary antiserum. The sections were finally mounted using VectaMount permanent mounting medium (Vector Laboratories, USA) or Vectashield plus 40, 6-diamidino-2-phenylindole (DAPI) mounting medium (Vector Laboratories). All images were observed using a Leica DMI4000 fluorescence microscope and captured with a DFC365FX camera (Leica, Germany). Positive cells were calculated manually.

Proteins were extracted from mouse tissues or cultured cells using RIPA buffer (Biocolor Ltd., Belfast, Northern Ireland, UK) as mentioned before [7 (link)]. The expression levels of proteins were quantified with ONE software (Bio-Rad Laboratories, Hercules, CA, USA) and normalized to that of β-actin. Then, the proteins were subjected to Western blot analyses (10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis; 50 μg protein per lane) employing rabbit anti-ABCA1 (Novus Biologicals, Littleton, CO, USA).

Alexa546-phalloidin, lipidTOX red, actin-GFP and carboxy-H₂DFFDA were from Thermo Fisher Scientific. AAPH was from Cayman Chemical. Rabbit anti-ABCA1 was from Novus Biologicals. Antibodies to active Caspase-3 and GAPDH were from Abcam. Filipin, mevinolin, N-acetyl-l-cysteine (NAC), MβCD, water-soluble cholesterol (CHOL) and all other chemicals were from Sigma. KRBH buffer (128.8 mM NaCl, 4.8 mM KCl, 1.2 mM KH₂PO₄, 1.2 mM MgSO₄, 2.5 mM CaCl₂, 5 mM NaHCO₃, 10 mM HEPE, pH 7.4) was used for all experiments.