Abc detection ihc kit

Human ovarian teratoma tissue sections and breast cancer tissue sections were prepared by Shenzhen Second People’s Hospital, and proved by the Ethics Committee of Shenzhen Second People’s Hospital in accordance with the principles of the 1964 Helsinki declaration. Immunohistochemistry (IHC) was performed using horseradish peroxidase/3,3′-diaminobenzidine (DAB) (ABC) detection IHC Kit (ab64261, abcam). Briefly, The slides were heated at 60 °C for 90 min, deparaffinized in xylene, rehydrated with 100%, 90%, 80% and 70% ethanol, and immersed in methanol with 3% H₂O₂ (hydrogen peroxide) for 10 min at room temperature to inactivate endogenous peroxidase. Antigens were heat-retrieved in sodium citrate buffer (10 mM sodium citrate and 0.05% Tween 20, pH 6.0) at 100 °C for 8 min. After blocked with 10 % serum for 30 min at 25 °C, the slides were incubated with primary antibodies against CDT2 (dilution 1:150) and DDB2 (dilution 1:150) overnight at 4 °C. Incubated with biotin-conjugated secondary antibody for 20 min at 25 °C, washed with PBS, and then incubated with streptavidin peroxidase for 15 min at room temperature, the sections were stained with DAB and counterstained in hematoxylin. After dehydration and coverslip, images were captured under microscope (Olympus IX73) using cellSens Dimension program.

The myometrium samples were embedded in paraffin and sliced into 5 μm thickness. To perform immunohistochemical staining, the Mouse and Rabbit Specific HRP/DAB (ABC) Detection IHC Kit (ab64264, Abcam) was used, following the manufacturer's instructions. Peroxidase quenching was used for the hydrogen peroxidase block. Tissue sections were incubated overnight in a moist chamber at 4°C with primary antibodies CD68 (1:500, ab201340, Abcam), CD3 (1:150, ab135372, Abcam), CD56 (1:20000, ab75813, Abcam), CD19 (1:100, ab134114, Abcam) or CD66 (1:100, ab197678, Abcam). After primary antibody incubation, the tissue sections were incubated with Biotinylated Goat Anti‐Polyvalent secondary antibody for 1 hour. The diaminobenzidine tetrahydrochloride (DAB) chromogen was used for the production of brown colouration. Images were captured by a Leica DMi8 fluorescence microscopy.

The mice were anesthetized by isoflurane inhalation and executed before harvesting the skin samples. The skin samples from the lesion area were fixed in 10% formaldehyde for 24 h and embedded in paraffin for sectioning. The skin sections were stained with hematoxylin and eosin (HE). The thickness of the epidermal, dermal, and various inflammatory cells was analyzed from HE-stained sections and visualized under a magnification of ×200. Five fields from each sample were randomly selected to measure the thickness of epidermis and dermis.
Immunoperoxidase staining was used to detect IL-33 and ST2-positive cells in the sensitized skin. Sections of 4 mm were prepared and stained by rabbit HRP-DAB (ABC detection IHC kit, Abcam). Briefly, endogenous peroxidase activity was blocked by peroxidase blocking solution and protein block was applied to exclude the nonspecific staining. Then, the sections were incubated with biotinylated goat polyclonal antibody, followed by streptavidin-peroxidase at room temperature. The sections were then stained in DAB substrate for 1 min. The tissue sections were mounted with Aquamount (BDH, Gurr, Pole, UK). The positively stained IL-33 (1.25 μg/mL; ab118503; Abcam) and ST2 (0.75 μg/mL; ab25877; Abcam) cells were counted in 10–15 high power fields (HPFs) at ×400 and expressed as cells/HPF, with mean and SE.