Abc reagent kit

All the paraffin tissue sections were dewaxed and rehydrated, and blocked with hydrogen peroxide (0.3% H₂O₂) for 30 min. Antigen retrieval was performed by heating the slides in sodium citrate buffer for 10 min. Immunohistochemistry analysis were performed using ABC reagent kit (Vector Laboratories, Burlingame, CA, United States). The slides were blocked with prediluted normal horse serum, followed by incubation with anti-drebrin (1:500 dilutions; Abcam, Cambridge, MA, United States) overnight at 4°C. After incubating with a secondary antibody of prediluted biotinylated horse anti-mouse Ig/rabbit Ig for 30 min and R.T.U ABC reagent for another 30 min at RT, DAB was used for color development, and then counterstained with hematoxylin. The stained sections were examined with an inverted microscope (DMi8, Leica, Wetzlar, Germany).

Sections (6 μm) were dewaxed with xylene (2 × 4 min), 100% (v/v) ethanol (4 × 1 min), 70% (v/v) ethanol (1 min) and dH₂O (1 min). Antigen retrieval was performed in 0.01 M sodium citrate buffer, pH 6.0 in a steamer for 30 min at 100 °C. Sections were blocked in 3% (v/v) normal goat serum (NGS) (Vector Laboratories, UK) in 0.05% (v/v) PBST for 1 h. Endothelial cells were stained with rabbit anti-CD31 (PECAM) primary antibody diluted 1:100 (Matrigel sections) or 1:200 (4T1.2luc tumours) in blocking buffer overnight at 4 °C. Non-specific rabbit IgG (Dako, Germany) antibody was used as isotype control. Biotin-conjugated goat anti-rabbit secondary antibody (Vector Laboratories, UK) in blocking buffer was incubated for 1 h. Antibody complexes were detected using the ABC reagent kit according to manufacturer’s instructions (Vector Laboratories, UK). Cell nuclei were counterstained with haematoxylin (VWR International, Ireland). Sections were dehydrated in 100% (v/v) ethanol (4 × 1 min) xylene (2 × 4 min) then mounted in DPX mounting medium (Sigma Aldrich, Ireland). CD31 positive endothelial cells were quantified in three fields of view (original magnification 200×) for each Matrigel implant (n = 5/group) or five fields of view (original magnification 400×) for each tumour (n = 3/group) and an average calculated for each group.

Thyroid tissue sections (5 μm‐thick) were stained by using the ABC reagent kit (Vector Laboratories). ITPR1 expression in normal and malignant samples was semi‐quantitatively scored.¹⁶ (link) The immunoreactivity was determined by the sum of the staining intensity and the area of positive staining according to negative (0), weak (1‐2), moderate (3), or strong (4‐6) staining.

Sections were fixed in methanol at − 20 °C, permeabilized with 0.5% Triton-X, incubated with 3% hydrogen peroxide to quench endogenous peroxidases and blocked in 5% goat serum for 1 h. Sections were then incubated overnight at 4 °C with either T22 for tau oligomers (1:200) or tyrosine hydroxylase antibody (Millipore) for dopamine (1:200). The following day, sections were washed in PBS three times for 10 min each and incubated with biotinylated goat anti-rabbit IgG (1:200; Vector Laboratories) for 1 h. Sections were then washed three times for 10 min each in PBS and processed using an ABC reagent kit (Vector Laboratories) and visualized with DAB substrate (Vector Laboratories) according to the manufacturer’s recommendations. Lastly, sections were counterstained with hematoxylin (Vector Laboratories) for nuclear staining and mounted. To quantify TH-positive cells, we acquired bright field images using a Nikon Multizoom AZ100 microscope equipped with a Nikon DS-2 M color CCD camera (Nikon Instruments Inc.). Three sections per mouse (n = 6-8 per treatment group) were imaged for quantification. Four visual fields from the olfactory bulb of each section were used to quantify TH-positive cell bodies using the ImageJ plugin, counting cell. Results were analyzed by one-way ANOVA with the Bonferroni post hoc test.