Goat serum

CD-1 mice were injected with LTR1.20-SFFV-GFP (2.34 × 10⁷ TU/mL) at post-natal day 1. Mice received vector either by direct intracranial injection into the left lateral ventricle (5 μL), or intravenously (20 μL). At 1 week later, they were sacrificed and organs imaged by fluorescence microscopy (Leica MZ16) or immunohistochemistry. For immunohistochemistry, brains were embedded in paraffin wax and sliced in the coronal plane in preparation for EGFP-staining. Brain sections were treated with 30% H₂O₂ in tris-buffered saline (TBS) for 30 min and blocked with 15% of goat serum (Vector Laboratories) in TBS-tween 20 (TBST) for 30 min. Rabbit anti-GFP (1:10,000; Abcam) was added in 10% goat serum in TBST and left on a gentle shaker overnight at 4°C. Goat anti-rabbit (1:1,000; Vector Laboratories) was then added in 10% goat serum in TBST for 2 hr. The sections were incubated for a further 2 hr with VECTASTAIN ABC (Vector Laboratories), followed by addition of 0.05% 3,3′-diaminobenzidine (DAB) and brief incubation. Sections were transferred to ice-cold TBS. Each individual brain section was mounted on chrome gelatin-coated Superfrost Plus slides (VWR) and left to dry for 24 hr. The slides were dehydrated in 100% ethanol and placed in Histo-Clear (National Diagnostics) for 5 min before mounting with DPX mounting medium (VWR).

Cells were seeded in 24-well culture plates and treated with IHT or transforming growth factor-β (TGF-β) (R&D Systems, Minneapolis, MN, USA) for 5 h. After washing with PBS, cells were fixed with 4% paraformaldehyde (Biosesang, Seongnam, South Korea) for 15 min. Cells were permeabilized with 0.1% Triton X-100 (Sigma-Aldrich, St. Louis, MO, USA,) for 15 min, and blocked with 10% goat serum (Vector Laboratories, Inc., Burlingame, CA, USA) for 15 min. Primary and secondary antibodies were prepared in 1% BSA in PBS and incubated for overnight or 2 h. The primary antibody used was anti-NR4A1 (1:100, #3960, Cell signaling Technology, Beverly, MA, USA), and secondary antibody used was Alexa Fluor^® 488 goat anti-rabbit IgG (1:50, A11008, Invitrogen, MA, USA). Pictures were taken under a LSM780 (Carl Zeiss, Oberkochen, Germany) confocal microscope.

Brain sections were washed with PBS for three times and were blocked with PBS-containing 3% goat serum (Vector Laboratories, #) and 0.1% Triton X-100 for 1 h at room temperature. Sections were incubated with primary antibodies overnight at 4°C and were washed with PBS. The following primary antibodies were used: GFAP (), mouse anti-Neuronal Nuclei (NeuN, Millipore, MAB377), DCX (), and GluA1 (Abcam, ab1232). On the second day, the samples were taken out and washed with PBS for 3 times, 5 min/time, followed by incubation with the secondary antibodies for 1 h at room temperature. Fluorophore-conjugated secondary antibody was used: goat anti-mouse Alexa Fluor 568 (Invitrogen, A11031), goat anti-rat Alexa Fluor 568 (Invitrogen, A11077), goat anti-rabbit Alexa Fluor 488 (Invitrogen, A11008), and goat anti-mouse Alexa Fluor 488 (Invitrogen, A11001). All the sections were observed and images were taken with a confocal microscope (Leica). The images were analyzed with Imarus software.

For immunohistochemistry the cryosections (5 μm) were fixed with ice-cold acetone (Merck) for 10 min. Non-specific binding was blocked using goat serum (Vector Laboratories) for 20 min. Primary antibodies, F4/80 BM8 and CD3 17A2 (Biolegend), were diluted in PBST and incubated overnight. For detection, ImmPRESS® HRP Goat Anti-Rat IgG Polymer Detection Kit was used with DAB (Dako) as a substrate. Images were obtained using Apotome (Zeiss) with camera for quantification.