Sigmafast dab peroxidase substrate

Animals were sacrificed by overdose of trifluoroethane and immediately perfused first with PBS (phosphate-buffered saline) and then with 4% paraformaldehyde (dissolved in PBS, pH 7.4). Brains were carefully removed and postfixed for 4 h in 4% paraformaldehyde (dissolved in PBS, pH 7.4). Tissue was cryoprotected using 30% sucrose/PBS, pH 7.4. Brains were immersed in Tissue-Tek® O.C.T. compound (VWR) and frozen at −80°C. Serial coronal sections (30 μm sections) were cut with a microtome. Free-floating sections were blocked with 4% goat serum/PBS plus 0.2% Triton X-100 and incubated with the primary antibody (anti-Grip1 antibody, BD Biosciences). Sections were sequentially incubated with biotin-conjugated anti-mouse (goat polyclonal; Jackson ImmunoResearch), ABC reagents (Vector Laboratories), and lastly SigmaFast DAB Peroxidase Substrate (Sigma) producing the dark brown reaction product in cells expressing Grip1.

Immunohistochemistry (IHC) and immunofluorescence (IF) was performed on 50 µm thick serial brain sections. Primary antibodies and working dilutions are detailed in Table S1. For histological studies, coronal sections were incubated in primary antibodies for P-α-syn or Tyrosine Hydroxylase followed by incubation with biotin-conjugated anti-mouse or anti-Rabbit antibody respectively (Vector Labs, Burlingame, CA, USA), ABC reagents (Vector Labs, Burlingame, CA, USA), and SigmaFast DAB Peroxidase Substrate (Sigma-Aldrich, St. Louis, MO, USA). Sections were counterstained with Nissl (0.09% thionin). Immunoreactivity in double-labeled sections was labeled using appropriate fluorescent secondary antibodies conjugated to Alexa-fluor 488, 594 or 647 (Invitrogen, Carlsbad, CA, USA). Images (IHC) were captured on AxioCam Mrc camera connected to Observer. Z1 microscope (Zeiss, Oberkochen, Germany). Images (IF) were obtained by confocal scanning microscopy (LSM710, Zeiss, Dublin, CA, USA). Photoshop CS6 (Adobe Systems) was used to assemble montages. Fine mapping of P-α-syn pathology was performed by tracing all visible immunoreactive inclusions/cells and neurites at 10×magnification from sections at multiple rostrocaudal levels corresponding to coronal sections at approximately +1.92, +0.74, −0.82, and −2.30, and −3.52 mm relative to Bregma.

Mice were perfused with PBS and 4% PFA and brains were removed, followed by fixation in 4% PFA overnight and transfer to 30% sucrose for cryoprotection. Immunohistochemistry (IHC) was performed on 40 μm thick serial brain sections. For histological studies, free-floating sections were blocked with 10% goat serum in PBS with 0.2% Triton X-100 and incubated with TH antibody followed by incubation with biotin-conjugated anti-rabbit antibody. After three times of washing, ABC reagent (Vector laboratories, Burlingame, CA) was added, and the sections were developed using SigmaFast DAB peroxidase substrate (Sigma-Aldrich). Sections were counterstained with Nissl (0.09% thionin). For the quantification, both TH- and Nissl-positive DA neurons from the SNpc region were counted by an investigator who was blind to genotypes or treatment condition with randomly allocated groups through optical fractionators, an unbiased method for cell counting, using a computer-assisted image analysis system consisting of an Axiophot photomicroscope (Carl Zeiss) equipped with a computer controlled motorized stage (Ludl Electronics, Hawthorne, NY), a Hitachi HV C20 camera, and Stereo Investigator software (MicroBright-Field, Williston, VT). The total number of TH-stained neurons and Nissl counts were analyzed as previously described (Kam et al., 2018 (link)).

Cryopreserved brains were frozen in OCT buffer. A microtome was used to cut 30 μm thick serial coronal sections. These were blocked with 4% goat serum plus 0.2% Triton X-100 in PBS and incubated with an antibody against TH (Novus Biologicals, Littleton, CO, USA). This was followed by incubation with biotin-conjugated anti-rabbit antibody (Vectastain Elite ABC Kit, Vector laboratories, Burlingame, CA, USA). The reaction color was developed using SigmaFast DAB Peroxidase Substrate (Sigma-Aldrich). Sections were counterstained with Nissl stain (0.09% thionin). TH and Nissl-positive neurons in the SNpc were counted using an unbiased stereological method as described previously (Kam et al., 2018 (link); Panicker et al., 2019 (link)) using a computer- assisted image analysis system consisting of an Axiophot photomicroscope (Carl Zeiss Vision) equipped with a computer controlled motorized stage (Ludl Electronics), a Hitachi HV C20 camera, and Stereo Investigator software (MicroBright-Field).

Mice were perfused with ice-cold phosphate buffered saline (PBS) followed by 4% paraformaldehyde/PBS (pH 7.4). Brains were removed and post-fixed for 4 hrs in the same fixative. After cryoprotection in 30% sucrose/PBS (pH 7.4), the brains were frozen and serial coronal sections (60 μm sections) were cut with a microtome. Free-floating 60 μm sections were blocked with 5 % goat serum/PBS plus 0.2% Triton X-100 and incubated with an antibody against TH (rabbit polyclonal; Novus Biologicals) followed by incubation with biotin-conjugated anti-rabbit antibody (anti-rabbit polyclonal; Vector Labs), ABC reagents (Vector Labs), and SigmaFast DAB Peroxidase Substrate (Sigma-Aldrich). Sections were counterstained with Nissl (0.09 % thionin).
As described before^{50 (link)}, TH-positive cells and Nissl from the SNpc and (Ventral tegmental area) VTA region were counted through optical fractionators, the unbiased, 3-diamensional method for cell counting. This method was carried out by using a computer-assisted image analysis system consisting of an Axiophot photomicroscope (Carl Zeiss Vision) equipped with a computer controlled motorized stage (Ludl Electronics), a Hitachi HV C20 camera, and Stereo Investigator software (MicroBright-Field). The total number of TH-stained neurons and Nissl counts were calculated.