Novared peroxidase substrate kit

Hematoxylin and eosin (H.E.) staining, Periodic Acid Schiff (PAS) staining, and immunohistochemistry were performed using paraffin embedded sections [71 (link)]. Primary antibodies with appropriate dilutions were used to detect target proteins (Table 1). An Avidin/Biotin Complex (ABC) protocol was used to amplify the immunoreactive signals, with the sequential addition of secondary biotinylated anti-rabbit antibody (BA-1000, Vector laboratories) or anti-rat (BA-9400) antibody and ABC reagents (Vector Laboratories, Burlingame, CA, USA). The NovaRED^™ Peroxidase Substrate Kit (Vector Laboratories) was utilized to develop signals for the targets. Quantitative analysis of uterine glands was carried out by counting the number of forkhead box A2 (FOXA2)-positive glands on the cross sections of control and Ezh2 Amhr2-Cre cKO mice. At least 11 sections from each uterine sample were analyzed. To determine differences in the abundance of perforin 1 (PRF1)-expressing cells between control and Ezh2 Amhr2-Cre cKO mice at E6.5 and E7.5, immunostaining was performed using anti-PRF1 antibody and the number of PRF1-positive cells quantified using the Qupath software [72 (link)].

Frozen sections from PS1-APP, WT littermates, and PS1-APP/CX3CR1^−/− or PS1-APP/CX3CR1^+/− were fixed in acetone for 2 min, washed in PBS, then treated with 0.25% trypsin for antigen retrieval. Endogenous peroxidase activity was quenched with 0.3% H₂O₂ followed by blocking with 1.5% donkey serum in PBS. Sections were incubated overnight at 25°C with rat anti-CD11b (clone 5C6) (AbD Serotec) or rat IgG2b negative control (AbD Serotec) each at 10 μg/ml in PBS with 1.5% donkey serum. The slides were then processed using the Vectastain® Elite ABC reagent (Vector laboratories) according to the manufacturer's instructions followed by development with the NovaRed™ Peroxidase Substrate kit. Aβ-containing plaques were stained with 1% Thioflavin-S (Sigma Aldrich) for 5 min in the dark. Finally, the sections were counterstained with hematoxylin, mounted with VectaMount and digitally photographed via brightfield microscopy to detect CD11b, and via fluorescence to visualize Thioflavin-S. Since different size plaques can exist within any single mouse at any single age, the size of the plaques was determined using Image J and the number of CD11b positive cells associated with plaques ≥75 μm in their largest diameter was quantified by two independent blinded laboratory members. A minimum of 80 plaques were counted per genotype.

Mature ovarian were fixed with Davidson’s fixative overnight, then embedded in paraffin solution. The paraffin sections of mature ovary (5-μm thick) were dewaxed with xylene, rehydrated with a decreasing grading alcohol, then treated with 1% hydrogen peroxide (H₂O₂) to quench any endogenous peroxidase activity. Subsequently, the sections were incubated with a blocking solution (2% BSA in PBS containing 0.4% Triton-X) for 30 min at room temperature, followed by 2 μg/ml (1:100) dilution of CTD110.6 antibody at 4 °C overnight. Thereafter, the sections were treated with 1:500 goat anti-mouse IgG conjugated with HRP (Abcam, Cambridge, UK) for 2 h at room temperature. The enzymatic product was visualized by a Nova Red peroxidase substrate kit (Vector laboratories, Burlingame, CA). Sections were counterstained with 0.1% hematoxylin and photographed using a DM3000 Leica microscopy.