Sp2 laser scanning confocal microscope

Double immunofluorescent labeling and confocal microscopy was performed on FFPE brain slides to detect cell types that exhibit 3α,5α-THP positive immunostaining. After deparaffinization and rehydration, antigen retrieval was performed using 10X Citra Plus buffer at pH 6.0 at 100 °C for 15 min. Slides were then rinsed in PBS and blocked for 1 hr with 10% donkey serum (Jackson Immunoresearch Labs Inc., West Grove, PA) and incubated in primary antibody for cell type specific markers: TH (1:1000; ImmunoStar) or NeuN (1:1000, (D4G40) XP, Cell Signaling Technologies) for 24 hr at 4°C. Next, slides were rinsed, blocked, and incubated with anti-3α,5α-THP primary antibody for 48 hr at 4°C. Then, sections were rinsed and incubated with secondary antibody (Alexa Fluor 488 for 3α,5α-THP visualization and Alexa Fluor 594 for cell-type specific markers; Life Technologies). Immunofluorescence was visualized using a Leica SP2 laser scanning confocal microscope and computer software.

Free floating sections (three to four sections/mouse) were rinsed, blocked in normal donkey serum, and incubated in primary antibody for vesicular transporter specific markers: for glutamate vesicles, vesicular glutamate transporter 1 (VGLUT1) [(1:1,000), Millipore Corporation, Billerica, MA, USA] and for GABA vesicles, vesicular GABA transporter (VGAT) [(1:500), Synaptic Systems, Goettingen, Germany] for 24 hours at 4 °C. Sections were then rinsed in PBS, blocked, and incubated with 3α,5α-THP primary antibody (1:500; purchased from Dr. R.H. Purdy) for 48 hours at 4 °C. Following 3a,5a-THP incubation, sections were rinsed and incubated with secondary antibody (Alexa Fluor 594, Life Technologies, Durham, NC, USA) for VGLUT1 and VGAT at 4 °C followed by rinsing and incubation with secondary antibody (Alexa Fluor 488) for 3a,5a-THP visualization. Immunofluorescence was visualized using a Leica SP2 laser scanning confocal microscope and computer software (Buffalo Grove, IL, USA). Vesicular transporter markers and 3α,5α-THP immunofluorescence were imaged sequentially to prevent fluorophore bleed-through. For image processing, average fluorescent intensity was calculated from 10 to 11 stacks per image using ImageJ software (National Institutes of Health, USA).