3 3 diaminobenzidine dab substrate kit

Neutral buffered formalin-fixed, paraffin-embedded brain tissue slices (5 μm thick) were used for immunohistochemical staining. The tissue slides were blocked with horse serum, incubated with an anti-PrP antibody (3F4), incubated with a biotinylated horse anti-mouse IgG secondary antibody using an ultrasensitive ABC peroxidase mouse IgG staining kit, and visualized with a 3,3′-diaminobenzidine (DAB) substrate kit (Thermo Fisher Scientific). After the sections were counterstained with hematoxylin, they were observed under a light microscope (BX51; Olympus, Southend-on-Sea, UK).

IHC was performed using conventional methods as previously described
[32] (link). Briefly, antigen retrieval was conducted using 10 mM sodium citrate buffer (pH 6.0) in a microwave oven for 15 min at 98°C, and endogenous peroxidase activity was quenched using 3% hydrogen peroxide (37°C for 10 min). After being blocked with normal horse serum, the sections were incubated with primary antibody at 4°C overnight, followed by incubation with horseradish peroxidase-conjugated secondary antibody at room temperature for 60 min. The slides were then processed with a 3,3′-diaminobenzidine (DAB) substrate kit (#3400; Thermo Fisher Scientific). Morphometric analyses were performed using ImageJ software in an operator-blind manner. For each sample, at least 5 randomly selected fields were analyzed, and the data were averaged.

Cells (0.1 × 10⁷) were cultured in a 6-well plate (Nunc) and incubated with and without rfhSP-D (20 µg/ml) in a serum-free DMEM-F12 for various time points. The cells were then mixed with 2× treatment buffer (50 mM Tris–HCL pH 6.8, 2% v/v β-merceptoethanol, 2% v/v SDS, 0.1% w/v bromophenol blue, and 10% v/v glycerol) and sonicated for 30 s before running on a SDS-PAGE (12% w/v) for 90 min at 120 V. The SDS-PAGE separated proteins were electrophoretically transferred onto a nitrocellulose membrane using an iBlot 7-min Blotting System (Thermo Fisher), followed by blocking with 5% w/v non-fat dried milk powder (Sigma) in 100 ml PBS for 2 h on a rotatory shaker at room temperature. The membrane was washed with PBST (PBS + 0.05% Tween 20) three times, each time for 10 min. The membrane was then incubated with primary anti-human TGF-β antibody, (1:1,000; R&D systems) at 4°C overnight on a rotatory shaker, followed by secondary anti-rabbit IgG horseradish peroxidase-conjugate (1:1,000; Promega) for 1 h at room temperature. The positive bands were visualized using 3,3′-diaminobenzidine (DAB) substrate kit (Thermo Fisher).