Th antibody

Mice were perfused with 4% paraformaldehyde, cryoprotected, and sectioned 30 μm-thick. Eight to twelve sections per brain were analyzed. Vectastain kit (Vector laboratories) was used to perform TH-staining according to manufacturer's directions using TH antibody (EMD-Millipore). Nissl staining was performed according to the manufacturer's protocol (IHCWORLD). TH-positive and Nissl-stained neurons in SNpc were counted by the image analysis tool of NIS-Elements AR microscope software. TH-positive striatal fibers were assessed by optical density. MPTP i.p. (intraperitoneal injection)-administered mice that were used for immunohistochemistry were 2 months old and received MPTP (30 mg kg⁻¹ free base MPTP) daily for six consecutive days (Tatton and Kish, 1997 (link); Jackson-Lewis and Przedborski, 2007 (link)).

Free-floating 35 μm coronal sections containing the SNpc were cut on a horizontal sliding microtome. A total of 8 sections were sampled at 105 µm intervals for each brain region. The free-floating sections were immune-blocked with 4% goat serum in 0.25% Triton/PBS for 2 h and then incubated with Iba-1 antibody (1:2000, Wako Chemicals, Richmond, VA) overnight at 4 °C. On the second day, the sections were washed by 1% BSA in 0.25% Triton/PBS before the incubation with anti-tyrosine hydroxylase (TH) antibody (1:2000, EMD Millipore, Temecula, CA) overnight at 4 °C. The double-label immunofluorescence pictures were taken under the confocal microscope by using Alexa-488 (green) and Alexa-594 (red) conjugated secondary antibodies (1:1000) to visualize the TH immune reactive (THir) or Iba-1 positive cells. Stereological counts of TH+ SNpc neurons were estimated using an optical fractionator method on an Olympus BX50 stereological microscope within user-defined boundaries. Samples were countered in a double-blind manner. Data were expressed as percentage to saline-injected controls [26 (link), 39 (link)].

Brains from newborn mice were fixed for 8 h in Immunofix and clarified with the X-Clarity system (Logos Biosystem, South Korea). Briefly, whole brains were polymerized with acrylamide “hydrogel solution” for 3 h at 37 °C with a vacuum followed by the passive clarification for 48 h at 37 °C in “clearing solution”.
Brains were then subjected to deep-tissue TH immunolabeling by staining with TH antibody (dilution 1:100, Merck Millipore) in PBS/0.3% Triton for 3 days, 1 day of washing followed by incubation with anti-rabbit Alexa 488 for 3 days (Jackson Immunoresearch).
Finally, brains were observed by a two-photon upright LSM 880 Zeiss microscope equipped by W Plan-Apochromat 20x/1.0 DIC (UV) VIS-IR M27 75 mm objective. The fluorochrome was excited with the IR laser set at 860 nm. Z-stack were set with the height of the sections scanning = ~1 μm. Images were then reconstructed using Zen lite 2.3 (Carl Zeiss).

Prosaposin PS18 was purchased from Elabscience (Texas, USA). Bovine serum albumin, fetal bovine serum, L-glutamate, NMDA, paraformaldehyde, polyethyleneimine, and Triton X-100, were purchased from the Sigma (St. Louis, USA). Alexa Fluor 488 (secondary antibody), B27 supplement, Dulbecco’s modified Eagle’s medium, Neurobasal Medium, and trypsin were purchased from Invitrogen (Carlsbad, USA). TH antibody was purchased from Millipore (Burlington, USA). In Situ Cell Death Detection Kit was purchased from Roche (Indianapolis, USA).
Adult male and time-pregnant Sprague–Dawley rats were purchased from BioLASCO, Taiwan. The use of animals was approved by the Animal Research Committee of the National Health Research Institutes of Taiwan (NHRI-IACUC-109097-M1). All animal experiments were carried out in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals (NIH Publications No. 8023, revised 1978).

Paraffin on the selected slides was removed with xylene and rehydrated through graded ethanol. Heat-induced antigen retrieval (HIER) was carried out for sections in 0.001 M EDTA buffer, pH = 8.00 using a Biocare decloaker at 60 degree for 18 hours. The slides were then stained with Tyrosine Hydroxylase (TH) antibody (Millipore, Cat#: ab1542, 1–200) for 1 h at room temperature, the section incubated with Rabbit anti-Sheep secondary antibody (Thermofisher, Cat#: PI31240, 1–500), and the signal was detected using Leica Refine RED Kit (LeicaBiosystem, Cat#: DS9390) on Bond RX auto-Stainer.