Immunohistochemical Analysis of Paraffin-Embedded Human Brain Tissue

Paraffin-embedded human brain tissue sections (2 men and 1 woman for both LB positive and negative cases) were deparaffinized and hydrated through a series of graded ethanol solutions followed by PBS. The sections were incubated in Citrate buffer (10 mM Trisodium citrate, 0.05% Tween-20, pH6.0) at 90°C for 10 min for antigen retrieval. After washing in PBS, tissues were incubated in PBS-T (0.3% Triton X-100 in PBS) for 10 min. Tissues were blocked in 5% normal goat serum for 30 min and incubated in primary antibody at 4°C overnight. Immunostaining was visualized with biotinylated secondary, followed by avidin-biotin (Vectastain Elite Kit, Vector Labs), and 3,3’-diaminobenzidine reaction as previously described [43 (link)]. For immunofluorescence analysis, immunostaining was visualized with either fluorescent secondary (1:250 dilution; Alexa Fluor 488 or 594, Molecular Probes). For immunofluorescence staining of cultured cells, cells were grown and transfected on chamber slides. After antigen retrieval, cells were stained using same methods as tissue staining. Slides were scanned using an AperioScan Scope CS (40×magnification; AperioTechnologies Inc.) and fluorescence images of representative areas were acquired on EVOS FL Digital Microscope (EMS), Olympus IX81-DSU Confocal Microscope (Olympus), or TCS SP2 AOBS Spectral Confocal Microscope (Leica).