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Brains were immersion-fixed in 10% formalin, then dehydrated and embedded in paraffin. Serial 10 µm-thick sagittal sections were cut using a microtome. For Aβ staining, brain slices were previously treated with 85% formic acid for 5 min at room temperature for antigen retrieval followed by a blocking step with 3% H₂O₂/10% methanol solution in PBS. Then, brain sections were incubated overnight with mouse anti-Aβ 4G8 antibody (Biolegend, Cat#800710) diluted 1:1,000 in PBS/0.02% Triton-X 100 (Sigma, St. Louis, MO, USA) and then incubated for 1 h with sheep anti-mouse IgG–Horseradish Peroxidase (HRP) secondary antibody (GE Healthcare, Little Chalfont, UK) diluted at a 1:500 ratio. Peroxidase reaction was visualized using 3,3′-Diaminobenzidine (DAB) Kit (Vector Laboratories, Burlingame, CA, USA) following manufacturer’s instructions. Stained sections were dehydrated in graded ethanol, cleared in xylene, and cover-slipped with DPX mounting medium (Innogenex, San Ramon, CA, USA). Brain tissue slices from an AD patient were counterstained with hematoxylin. ThS staining was performed in brain slices consecutive to 4G8 stained sections. For this, tissue slices were incubated with a ThS (Sigma, St. Louis, MO, USA) solution (0.1% ThS in 50% ethanol) for 15 min after deparaffinization. After incubation, sections were washed for 2 min in 80% ethanol, dehydrated and cover-slipped.

To assess dopaminergic axonal and cell body degeneration in the right striatum and SNc, respectively, 6OHDA injected rats underwent transcardiac perfusion with 4% paraformaldehyde and heparin and 24-hour postfixed with 4% paraformaldehyde. Brains were cryoprotected with 30% sucrose and free-floating 60μm sections containing the striatum and SNc were collected and placed in phosphate-buffer solution for tyrosine hydroxylase (TH) immuno-reactivity. In brief, sections were processed in primary antiTH antibodies (Santa Cruz Biotechnology, rabbit IgG CAT# sc-14007), treated with peroxidase conjugated goat anti-rabbit secondary antibodies (Jackson Immunoresearch, West Grove, PA) visualized using a 3,3′diaminobenzidine (DAB) kit (Vector labs, Burlingame, CA) and mounted onto slides (Superfrost Gold Plus, Fisher Scientific) for image quantification. TH density was calculated for both hemispheres in the striatum and SNc using ImageJ (ver1.47, NIH, MD, USA). After, values from regions of interest in the lesioned right side were divided by coinciding regions of interest in the contralateral intact left side to quantify percent TH depletion (Rasband, 2014 ). Brain sections containing the STN were cut at 60 μm and collected on slides for cresyl violet staining to confirm correct electrode placement in this area (Figure 2C) using a rat atlas (Paxinos, 1998 ).

Under anesthesia, the control and DcKO mice at postnatal one and three months were perfused from the ascending aorta with 4% formaldehyde in 0.1 M phosphate-buffered saline. The mandibles were dissected and further soaked in the same fixative for 48 hours, followed by demineralization in 14% EDTA (pH 7.4) at 4C for 2 weeks. The tissues were processed for paraffin embedding, and serial 5 µm sections were prepared. The sections were stained with hematoxylin and eosin (H&E) for histological analyses. Picrosirius Red staining (Junqueira, Bignolas, & Brentani, 1979 (link)) was performed to assess the morphology and organization of the collagen fibrils.
For the immunohistochemistry analyses, anti-DSP-2C12.3 monoclonal antibody was used at a concentration of 2.05 µg/ml. Anti-DMP1 monoclonal antibody that recognizes the C-terminal region of DMP1 was used at a concentration of 4.7 µg/ml. Anti-BSP monoclonal antibody 10D9.2 was used at a concentration of 4.5 µg/ml.
All the IHC experiments were carried out using the mouse on mouse kit for monoclonal antibodies (Vector Laboratories, Burlingame, CA). The 3, 3′-diaminobenzidine (DAB) kit (Vector Laboratories) was used for color development according to the manufacturer’s instructions. Methyl Green was used as the counterstain.