Cy3 conjugated streptavidin

Constructed biotin-labeled miR-325-3p (Sangon Biotech, China) for fluorescent miRNA in situ hybridization was applied as previously described [30 (link)]. Briefly, after decalcification, cryo-cross-sectioned sections were cut into 10-μm thick slides. Then, after dewaxing and rehydrating, the slides were incubated with proteinase K (20 μg/ml, Ambion, USA), followed by prehybridization for 1 h under 37 °C temperature. After removing the prehybridization buffer, the miR-325-3p-biotin probe (5′-UUGAUAGGAGGUGCUCAAUAAA-3′-biotin) was incubated at 60 °C overnight. The following day the slides were incubated with a blocking agent (3% BSA in 0.1% PBST) for 1 h at room temperature after washing with 2 × SSC, 1 × SSC, and 0.5 × SSC, sequentially. Then the slides were incubated with Cy3-conjugated streptavidin (Jackson ImmunoResearch, USA) for 1 h. DAPI was used to label the cell nucleus. Images were captured using an Axio Imager one microscope (Zeiss, Oberkochen, Germany). For quantitative analysis, six independent sections in each group were chosen to evaluate the relative miR-325-3p positive cell ratio using Image J software.

Microarray slides were incubated with purified antibody (20 (link)). Purified IgG (1 μg/ml) and purified IgA (5 μg/ml) were diluted 1/10 in SuperBlock T20 (Tris-buffered saline [TBS]) blocking buffer (Thermo Scientific) and incubated for 1 h at 30°C with the peptide microarray slide. The slides were then washed with 5 ml of TBS buffer–0.1% Tween 20 for 3 min on a shaker at room temperature for 5 washes. Next, the slides were placed in the individual chambers of a Sarstedt Quadriperm dish and incubated with Alexa Fluor 647-conjugated AffiniPure mouse anti-human IgG (H+L) (Jackson ImmunoResearch Laboratories Inc.) and biotin-conjugated anti-monkey IgA (Alpha Diagnostics International) for 1 h at room temperature. The slides were then washed 5 times with TBS buffer–0.1% Tween 20. Cy3-conjugated streptavidin (Jackson ImmunoResearch Laboratories Inc) was added to the slide, and the slide was incubated in the dark for 1 h at room temperature. The slides were then washed 5 times with TBS buffer–0.1% Tween 20 and 5 times with deionized water. To dry, the slides were placed in a 50-ml Falcon tube and spun at 1,400 rpm for 5 min. A control slide incubated with secondary antibodies alone without sample was also run to determine the background.

For Neurobiotin labeling, moths were restrained by taping the thorax tightly to a holder to prevent movement of the flight muscles. The head and thorax were fixed with wax, and the head capsule was opened to expose the brain. Neurobiotin (Vector Laboratories, Burlingame, UK) crystals were applied to the tip of a borosilicate micropipette, which was inserted manually into the target brain region. The brain was dissected and fixed overnight at 4°C in a fixative containing 4% PFA, 0.25% glutaraldehyde, and 2% saturated picric acid (in 0.1 M phosphate buffer). Brains were washed 4 × 15 min in 0.1M PBS, bleached in 10% hydrogen peroxide for either 30 min, 1 and 2 h, washed in Tris-HCl 3 × 10 min, and then incubated with Cy3-conjugated streptavidin (1:1000; Jackson ImmunoResearch, West Grove, PA, USA; catalog number 016-160-084) for three days at 4°C. After incubation, brains were rinsed 6 × 15 min in PBT and 2 × 20 min in PBS, dehydrated in an ascending ethanol series (50, 70, 90, 95 and 100%; 15 min each), treated with a 1:1 mix of 100% ethanol and methyl salycilate for 15 min, and eventually cleared for at least 45 min in pure methyl salycitate. As for antibody labeled preparations, brains were mounted in Permount between two coverslips, using plastic spacers to prevent squeezing of the brains.

At the end of each experiment, mice were sacrificed with a lethal dose of sodium pentobarbital and perfused with a solution containing 4% paraformaldehyde in 0.1 M phosphate buffer (PB, pH 7.4). Brains were extracted and stored in PBS solution until the cutting. Before cutting, brains were transferred into PBS containing 30% sucrose for at least 48 hr. Coronal slices (25 μm thick) containing the entire striatum from the recorded side (from AP 1.4 mm to AP −1.3 mm, following Paxinos and Franklin, 2001 ), were obtained using a digital automatic cryotome and collected on gelatine coated slides. Sections were incubated over night with Cy3-conjugated streptavidin (Jackson Immuno Research Laboratories) diluted (1:1000) in 1% BSA, 0.3% Triton-X 100 in 0.1 M PBS. Finally, the glass slides were covered with mowiol (Calbiochem) and imaged. Neurons were then reconstructed using a fluorescence microscope (DM 6000B, Leica) and a camera (DC350 FX, Leica) and then processed by ImageJ.

After recordings, slices were placed in a fixative containing 4% FA and 0.2% picric acid in 0.1 M PB (pH = 7.4) for 12 hr at 4°C. They then were embedded in agarose (2%) and re-sectioned at ~150 µm thickness. The biocytin-filled cells were visualized with Cy3-conjugated streptavidin (1:1000, Jackson ImmunoResearch, Bar Harbor, ME) in TBS containing 0.2% Triton X-100. Sections were then treated with uranyl acetate, dehydrated in a graded series of ethanol, incubated in acetonitrile, and flat-embedded in epoxy resin (Durcupan) as described in Holderith et al., 2020 (link). Putative contacts between the recorded neurons were identified with visual inspection at high magnification (60×, 1.35 NA objective, Olympus FV1000 microscope, Tokyo, Japan). Tissue blocks containing the biocytin-filled processes were re-embedded, and ultrathin (70 or 200 nm) serial sections were cut and mounted on adhesive Superfrost Ultra plus slides. Potential contact sites between the presynaptic PC boutons, and the postsynaptic dendrites were identified on the ultrathin sections, imaged using a confocal microscope (Olympus FV1000), and reconstructed with a custom-made ImageJ plugin (HyperStackStitcher, 3DHistech, available on the website: http://www.nusserlab.hu/software.html).

The terminal deoxynucleotidyl transferase-mediated dUTP–biotin nick end labeling (TUNEL) technique was employed to detect apoptotic cells, as previously described [40 (link)]. Tissue slices were washed with PBS (3 × 10 min) and treated with a cold solution of ethanol–acetic acid (2:1) for 5 min. Then, slices were washed with PBS (3 × 10 min) and permeabilized with 0.2% v/v Triton X-100 and 0.1% w/v sodium citrate in distilled water for 15 min. They were again washed with PBS (2 × 10 min) and incubated with TUNEL buffer containing 30 mM Tris–HCl, 140 mM sodium cacodylate, 1 mM cobalt(II) chloride, and 0.3% v/v Triton X-100 for 30 min. Subsequently, the tissue slices were incubated in a medium with terminal transferase (800 U/ml; Roche, Basel, Switzerland) and biotinylated dUTP (1 µM; Roche) in TUNEL buffer for 1 h and 30 min at 37 ºC. The reaction was terminated by adding saline sodium citrate buffer, composed of 150 mM sodium chloride and 15 mM sodium citrate in distilled water. Finally, the slices were washed with PBS (3 × 10 min) and developed with a medium containing Cy3-conjugated streptavidin (1:200; Jackson ImmunoResearch Laboratories) in PBS for 1 h and 30 min. Both nuclear counterstaining and slide mounting were performed as described above.

For Pax7 immunostaining, fresh sections were fixed in 4% paraformaldehyde for 20 min, permeabilized with methanol (-20°C) for 6 min, and blocked first with a solution containing 4% bovine serum albumen (BSA; Jackson) in PBS and then with goat anti-mouse AffiniPure Fab fragment (1:100; Jackson) after antigen retrieval with 100 mmol/L sodium citrate. The sections were then incubated overnight at 4°C with an anti-Pax7 antibody (1:20; Developmental Studies Hybridoma Bank). After washing with PBS, Pax7 signals were visualized by incubating with biotin-conjugated goat anti-mouse IgG₁ (1:1000; Jackson) and Cy3-conjugated streptavidin (Jackson; 1:2500). Nuclei were stained with 4’,6-diamidino-2-phenylindole (DAPI; Invitrogen). For MyoD immunostaining, the fresh sections were fixed in 4% paraformaldehyde for 20 min, permeabilized with 0.2% Triton X-100/PBS (PBST) for 10 min, and blocked by incubating with 5% BSA/6% goat serum/0.2% PBST at room temperature for 2 h. Immunostaining with anti-MyoD antibody (1:50; Santa Cruz) was performed by overnight incubation at 4°C. After washing, immunoreactive proteins were visualized by incubating with fluorescein isothiocyanate (FITC)-conjugated goat anti-rabbit IgG. Nuclei were stained with DAPI (Roche). The Pax7⁺ and MyoD⁺ cells in the sections (n = 10) were counted.