Biotinylated goat anti mouse secondary antibody

Lumbar or sacral mouse or rat spinal cord sections were used for preembedding immunoperoxidase labeling of VGLUT3. Mouse tissue sections were initially incubated in 1% NaBH₄ in PBS for 30 min to quench free aldehyde groups. After permeabilization in 50% ethanol for 30 min and incubation in PBS with 1% BSA for 1 h, the sections were incubated in primary antibody solution (mouse anti-VGLUT3, 1:1000 in PBS with 1% BSA) at room temperature overnight or for 72 h. After rinsing, the sections were subsequently incubated in biotinylated goat anti-mouse secondary antibody (Vector Laboratories) in PBS with 1% BSA and in Vector ABC (Vector Laboratories) for 2–3 h each. Peroxidase activity was visualized by incubation in ImmPACT DAB (Vector Laboratories) for 30 s to 2 min. Sections thus labeled for VGLUT3 were rinsed briefly in PB (0.1 M; pH 7.4), incubated in 0.5–1% OsO₄ in PB for 10–30 min (depending on section thickness), dehydrated in graded series of ethanol and embedded in Durcupan (Electron Microscopy Sciences). Ultrathin sections of embedded tissue were counterstained using 2% uranyl acetate (15 min) or UranyLess (2 min; Electron Microscopy Sciences) followed by 0.4% lead citrate before examination in a JEOL 1230 electron microscope.

Immunohistochemical staining was performed as described below. Reagents used in this study were described as follows: mouse anti-M1 monoclonal antibody (1:200 dilution, a gift from Professor Wenjun Liu, Institute of Microbiology, CAS.), biotinylated goat-anti-mouse secondary antibody (1:200 dilution, Vector Laboratories, USA), and strepavidin-conjugated HRP (1:200 dilution, Jackson Immuno Research, West Grove, PA).

Rats were deeply anesthetized with sodium pentobarbital (60 mg/kg) and perfused with 100 mL of ice-cold 0.9% saline followed by ~250 mL of 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4). Brains were postfixed overnight then transferred to 30% sucrose and stored at 4°C until sectioning. 30 μm sections were obtained in a −20°C cryostat and stored at 4°C in cryoprotectant until staining. Phosphorylated ERK1/2 immunoreactivity was detected by incubating sections with mouse anti-p44/42 MAPK (1:200, 48 h, 4°C, Santa Cruz Biotechnology #SC-7383, Dallas, TX) and biotinylated goat anti-mouse secondary antibody (1:200, 90 min, 4°C, Vector Labs, #BA-9200, Burlingame, CA). Phosphorylated ERK1/2 was visualized with the avidin-biotin horseradish peroxidase method (90 min, 4°C, Vectastain Elite ABC Kit, Vector Labs) and nickel-enhanced 3,3′-diaminobenzidine (Sigma-Aldrich) as chromogen for 10 min at room temperature. One left and one right hemisphere section corresponding to Bregma +2.7 mm were counted for each subject. The rostrocaudal level was determined by comparing an adjacent section stained with cresyl violet to the stereotaxic atlas (Paxinos and Watson, 1998 ). One observer, blind to group membership, quantified phosphorylated ERK-stained neurons that included a clearly stained cell body and least one dendritic process as shown in Figure 3.

The IHC staining was done as previously described [24 (link)]. Mouse on mouse blocking reagent for vimentin staining, ABC-HRP kit and ABC-AP kit were purchased from Vector Laboratories (Burlingame, CA). CD31 antibody (Thermo Fisher Scientific, Reinach, Switzerland) and biotinylated goat anti-rabbit secondary antibody (Vector Laboratories) were diluted 1:50 and 1:200, respectively. Vimentin antibody and biotinylated goat anti-mouse secondary antibody (Vector Laboratories) were diluted 1:200 and 1:800, respectively. CYR61 antibody (Santa Cruz Biotechnology, Inc., Heidelberg, Germany, #sc13100) and biotinylated goat anti-rabbit secondary antibody were diluted 1:150 and 1:600, respectively. For CD31/vimentin double-staining, tissues were first stained with anti-CD31 and biotinylated goat anti-rabbit secondary antibody, followed by anti-vimentin-staining and biotinylated goat anti-mouse secondary antibody. Substrates used were DAB (Sigma-Aldrich) and Vector Red (Vector Laboratory).

To assess dopaminergic neuronal survival, we performed TH-positive (TH⁺) cell counting as described previously [27 (link), 29 (link)]. First, the sections were incubated with a blocking solution (0.1% Triton X-100 and 10% FBS in PBS), then the endogenous peroxidases were inhibited with H₂O₂ and later incubated overnight at 4 °C with the primary antibody mouse anti-TH (1:500, Transduction Laboratories). After several rinses, the slices were incubated with biotinylated goat anti-mouse secondary antibody (1:200, Vector Laboratories) for 1 h at room temperature. Subsequently, the Vectastain ABC kit was added, and the resulting product was visualized by adding DAB to the slices until color developed (5–10 min). Afterward, the sections were counterstained with Nissl (0.25% Cresyl Violet dissolved in acetate buffer) for 4 min, washed in tap water, air-dried, cleaned with xylene, and mounted with Entellan™ (Merck).
Quantification of the TH⁺ neurons in the mice SN was performed in five consecutive coronal sections separated by 240 µm. The SN was carefully delineated, and the number of TH⁺ cells in each condition was counted. Images were acquired under the magnification of 10 × at the Zeiss Axiovert 200 imaging microscope (Axiobserver Z1, Zeiss), and the number of TH⁺ cells was counted using the ImageJ program.

To visually confirm the success of 6-OHDA infusion, immunostaining for tyrosine hydroxylase (TH) was performed at the striatum and substantia nigra [40 (link)]. All experimental animals were perfused with 4% paraformaldehyde and then, brains were postfixed in the perfusion solution and cryoprotected in 25–30% sucrose. Coronal sections 40 μM thick were prepared with a freezing microtome and serial sections were processed immunohistochemically. Staining for TH occurred by incubation of a monoclonal mouse anti-TH antibody (Sigma-Aldrich; #T1299, 1:250), followed by incubation of a biotinylated goat anti-mouse secondary antibody (Vector Laboratories, Burlingame, California, USA, 1:200). Sections were amplified with the avidin-biotin complex method (Vectastain Elite ABC‐HRP Kit; Vector Laboratories) for 1 and detected by the 3,3’-diaminobenzidine method (Sigma-Aldrich). This qualitative verification of 6-OHDA infusion by TH staining has been previously described in detail by our group [40 (link)].

IHC was conducted as previously described [15 (link), 49 (link), 87 (link)]. Antigen retrieval for BRM was performed with 10mM Tris (pH 10) according to the manufacturer's guidelines. The anti-BRM rabbit antibody is described in Glaros et al. 2007 [49 (link)]. A goat anti-rabbit or goat anti-mouse biotinylated secondary antibody was used with these primary antibodies at 1:200 (Vector Labs). The sections were incubated with primary antibodies for 2 hours at room temperature and with secondary antibodies for 1 hour. We used an ABC staining kit with DAB/nickel detection reagent (BD Pharmingen, San Diego, CA, USA). Slides were counterstained with Harris hematoxylin for 2 minutes.