Ultrasensitive sp mouse rabbit ihc kit

Ninety epithelial OC (EOC) tissues including High-grade serous adenocarcinoma, low-grade serous adenocarcinoma, clear cell adenocarcinoma, mucinous cystadenocarcinoma, endometrioid adenocarcinoma and carcinosarcoma, were obtained from the Department of Pathology, First Affiliated Hospital, Gannan Medical University (Supplementary Tables 3 and 4). All tissues were examined by specialists using the World Health Organization criteria. Determination of OC stage and grade was according to the International Federation of Gynecology and obstetrics. The use of these specimens and patient’s information was approved by the Ethics Committee of First Affiliated Hospital, Gannan Medical University. The OC CDXs and PDXs xenograft tumor tissues were fixed with formalin and Paraffin-embedded. H&E staining was performed using a standard histological protocol. OC tissues were collected and made into 4-μm-thick paraffin tissue microarrays for immunohistochemistry (IHC) staining. The staining procedure was carried out using the UltraSensitive™ SP (mouse/rabbit) IHC Kit and DAB Kit (MXB, Fuzhou, China) according to the standard procedure. Imaging was captured using the TissueFAXS Plus system (version 7.0, TissueGnostics GmbH, Vienna, Austria).

After the behavioral tests, the anesthetized rodents were transcardially perfused with PBS and then 4% paraformaldehyde, and the brain was harvested. The brain was fixed in 4% paraformaldehyde and embedded in paraffin to prepare sections with a thickness of 4 μm. IHC was performed according to the instructions of the UltraSensitive SP (Mouse/Rabbit) IHC Kit (KIT-9720, MXB Biotechnologies, Fujian, China). After deparaffinization and rehydration, the slices were immersed in antigen retrieval solution, heated in a microwave oven for 15 min, and then washed with PBS three times after 2 h of natural cooling. The slices were incubated in 3% H₂O₂ for 15 min to eliminate the effect of endogenous hyperoxide and then washed three times with PBS solution. Next, the slides were blocked with blocking buffer at room temperature for 1 h and then incubated with primary antibodies (Iba1, 1:200, 10,904–1-AP, Proteintech) overnight at 4 °C. The peroxidase-labeled polymer secondary antibody was added dropwise and incubated for 1 h at 37 °C, after which the slides were washed with PBS and then incubated with DAB (MXB biotechnology, Fujian, China) for 1 min at room temperature until a brown color developed. Finally, cover glasses were mounted onto glass slides with neutral resin and visualized using an optical microscope.

The mouse brain was fixed in 4% paraformaldehyde and dehydrated in 20–30% sucrose buffer for 3 days. The brains were embedded in OCT (Solarbio, Beijing, China) and then coronally sectioned at a thickness of 30 μm using a Leica cryostat. For immunohistochemical staining, UltraSensitive™ SP (mouse/rabbit) IHC Kit (MXB KIT-9720, Fuzhou, China) was employed, involving DAB staining and subsequent dehydration and clearing. Imaging was conducted by scanning the slices using an optical microscope (CS2, Leica Microsystems, Germany). For immunofluorescence experiments, brain slices or fixed-cultured cells were blocked to prevent non-specific binding and then incubated overnight at 4 °C with primary antibodies, followed by incubation with fluorescently labeled secondary antibodies at RT for 1 h. The nuclei were counterstained with DAPI for visualization. The slides were imaged using a laser scanning confocal microscope (SP8, Leica Microsystems, Germany). The fluorescence intensity in these images was quantified using ImageJ software.