Vector vip substrate kit

TG were harvested from control or Avil-SPP^-/- mice before or after infection, frozen in OCT compound and stored at -80°C until processing. Frozen tissue was sectioned using a MicroM HM 550 Cryostat. Tissue sections were fixed with 4% paraformaldehyde, washed with 1x phosphate-buffered saline (PBS), and then permeabilized with 0.3% Triton X-100 in PBS. Slides were blocked using 1x sea blocker (Thermo Scientific, Rockford, IL) plus 5% horse serum for 1 hr at 25°C, then incubated with anti-SPP antibody (Bethyl Laboratory, A304-404A) or anti-HSV-1 antibody (GeneTex, GTX26506) in blocking buffer at 4°C overnight. Slides were washed 3X with 1xPBS and incubated with biotin-conjugated secondary antibody for 2 hr at 25°C. Slides were washed 4X with 1xPBS and developed using a Vector VIP substrate kit according to company’s instructions (Vector Laboratories, Burlingame, CA. Cat# SK-4600). Slides were mounted with Permount mounting medium (Fisher Scientific, SP15-100) and image acquisition and data analysis were done using a Leica DM4000 microscope (Leica Microsystems, Buffalo Grove, IL). Density of the staining was calculated by dividing mean gray value by area of each image measured by Image J software [88 (link)].

For immunohistochemical analysis, 3 μm sections of F2 fish of 3 wpf, 5 fish per genotype (wt, npc1^+/−, npc1^−/−) of npc1^Δ56 and npc1^Δ7 lines were dewaxed and rehydrated at room temperature in a humid chamber. Slides were washed with 10 mM phosphate-buffered saline and 0.5% Tween 20 (pH 7.4) in three successive 5 min immersions. Endogenous peroxidase was quenched with Bloxall Blocking Solution (Vector Laboratories, Inc.; Burlingame, CA, United States) and the sections were incubated with a primary rabbit monoclonal recombinant anti-Niemann Pick C1 antibody [EPR5209] (ab134113 Abcam; Cambridge, United Kingdom) generated against the C-terminal region of the protein. Immunohistochemical staining was carried out with ImmPRESS HRP Horse Anti-Rabbit IgG Polymer Kit (Vector Laboratories, Inc.), together with the Vector VIP Substrate Kit (Vector Laboratories, Inc.) as chromogen. Negative controls were included in the experiment by using the antibody dilution solution without primary antibody. Sections were counterstained with hematoxylin.

Mice were perfused transcardially with 4% paraformaldehyde in 0.1 M PBS, pH 7.4. Brains and spinal cords were dissected and post-fixed overnight at 4°C, followed by cryo-protection in 20% sucrose in PBS for a minimum of 24 h. Coronal brain sections (40 μm) were cut using a VT1000 S vibratome (Leica). Transverse spinal cord sections (30 μm) were cut on a Leica SM2400 microtome (Leica Microsystems). Sections were stored at 4° C in PBS containing 0.1% sodium azide. Endogenous peroxidase activity was quenched by incubation in 0.3% H₂O₂ for 30 min. Following a brief wash in PBS + 0.1% Triton-X100 (PBST), the sections were incubated in blocking buffer (PBST + 5% normal goat serum) for 15 min. This was followed by an overnight incubation at room temperature with primary antibody in blocking buffer. After three rinses with PBST, the sections were incubated with biotin-conjugated secondary antibodies for 1 h at room temperature. Following a further three rinses with PBST, the avidin–biotin-conjugated complex was applied at room temperature for 30 min. The signal was visualised with the Vector VIP substrate kit (Vector Laboratories). Tissue sections were mounted on frosted end glass slides (Thermo Scientific) and coverslipped.

Eyes from Pax6-SPP^-/- and WT control mice at around two months of age were harvested, frozen in OCT compound, and stored at -80°C until further processing. Frozen tissue was sectioned using a Cryostar NX70 (Thermo Scientific, Rockford, IL). Tissue sections were fixed with 4% paraformaldehyde, washed with 1× phosphate-buffered saline (PBS), and then permeabilized with 0.3% Triton X-100 in PBS. The slides were blocked using 1× sea blocker (Thermo Scientific) plus 5% horse serum for 1 hr at 25°C, then incubated with anti-SPP antibody (Bethyl Laboratory, A304-404A, Waltham, MA) in blocking buffer at 4°C overnight. Slides were washed three times with 1XPBS and incubated with Biotin-conjugated secondary antibody for 2 hr at 25°C. Slides were then washed four times with 1xPBS and developed using a Vector VIP substrate kit according to company instruction (Vector Laboratories, Burlingame, CA. Cat# SK-4600). Vector Methyl green was used for counter staining. Slides were mounted with Permount mounting medium (Thermo Scientific, SP15-100) and image acquisition and data analysis were performed using a Leica DM4000 microscope (Leica Microsystems, Buffalo Grove, IL). SPP staining was quantified using Image J software.

Fly heads (2 μL/per head, 30 heads/blot) were homogenized in cold Tris buffer (25 mM Tris-HCl, pH 7.4, 15 mM NaCl, 1 mM EGTA, 1 mM ethylenediaminetetraacetic acid, supplemented with protease and phosphatase inhibitors) and centrifuged at 3000 × g for 3 minutes, followed by a 1 hour spin of the supernatant at 100,000 × g. Sarkosyl extraction, SDS-PAGE, and western blotting were carried out as described (Colodner and Feany, 2010 (link)). The primary anti-Tau antibodies were HT7 (1:500), Tau46 (1:500), AT270 (1:5000), AT8 (1:100), and AT180 (1:500). HT7 and Tau46 are phosphorylation-independent and specific for the amino- and carboxy-terminal regions of Tau, respectively. AT270, AT8, and AT180 are phosphorylation-dependent and recognize pT181 (AT270), pS202, pT205, and pS208 (AT8) (Malia et al., 2016 (link)), and pT231 (AT180) in Tau. The anti-beta actin antibody was used at 1:1000. Except for anti-beta actin (Abcam), all antibodies were from Thermo Scientific Pierce. For immunohistochemistry, paraffin-embedded brain sections (4 μm) were incubated with primary antibody (HT7, 1:400; AT8, 1:400, MC-1, 1:100) for 24 h at 4 °C, washed, incubated with secondary antibody, and the signal visualized using a Vector VIP substrate kit (Vector Laboratories) with an Olympus BX41 microscope equipped with an integrated 3.0 megapixel CMOS camera.

Mice were terminally anaesthetised and transcardially perfused with 20 ml cold PBS, followed by 20 ml 4% paraformaldehyde in 0.1 M phosphate buffer. Brains and spinal cords were dissected and postfixed overnight. Fixed tissues were paraffin-embedded and 8 μm sections cut. Following deparaffinisation, the sections were incubated in blocking buffer [PBS + 0.1% Triton X-100 (PBST) + 10% foetal calf serum] for 15 min at room temperature, followed by an overnight incubation with primary antibody specific for pS129 α-synuclein (ab51253, Abcam, 1:5000 dilution) in blocking buffer. After three rinses with PBST, the sections were incubated with biotin-conjugated secondary anti-rabbit antibody (1:200 dilution) for 1 h at room temperature. The antigen was visualised with the Vector VIP substrate kit (Vector Laboratories). Fixed, deparaffinised tissue sections were stained using Campbell-Switzer [4 (link), 5 ] or Gallyas [6 (link), 15 ] silver, as described. All sections were counterstained with haematoxylin and coverslipped using Pertex mounting medium.