3 3 diaminobenzidine (dab)

Immunohistochemistry and immunofluorescent staining was performed using an affinity-purified anti-EGR-1 and anti-pERK1/2 antibodies. The empirical method was identical as previously described [20 (link)]. After dewaxing, rehydration and washing, antigen retrieval was operated in EDTA buffer solution, and the endogenous peroxidase activity was inhibited by incubation with 3% H₂O₂ for 30 min. Then the sections were incubated with 5% normal fetal bovine serum at room temperature for 30 min. Then primary antibodies (EGR-1, 1:200; pERK1/2, 1:100) were loaded to brain slices (4°C, 12 h). After washing with PBS, peroxidase activity was developed using DAB as the chromogen (Maixin, Fuzhou, China).
The experimental results were photographed under a confocal microscope (×400). Nuclei were counterstained with DAPI, and the stained cells were analyzed and photographed under a confocal microscope(×400).

Using immunohistochemistry (IHC), we evaluated the expression of CXCL10 in goat uterus. Slices were taken out of the refrigerator at 4 °C and baked for 30 min at 60 °C. To remove the paraffin from the slices, they were submerged in xylene and a succession of alcohol solutions of varying concentrations. Slices were placed in PH6.0 citrate buffer, heated to a boil in a microwave, and then cooked for 15 min on low heat. The sample was then washed three times with PBS for five minutes each after cooling to room temperature. Histochemical staining was carried out using the ready-to-use immunohistochemistry hypersensitivity UltraSensitive^TM SP Kit (Maixin-Biotech, Fuzhou, China). The negative control section was switched out for PBS, and the primary antibody CXCL10 (Abbexa, Cambridge, UK) was incubated at a 1:300 dilution. Hematoxylin was counterstained for 25 s while the DAB (Maixin-Biotech, Fuzhou, China) color development time was monitored under a microscope. After being exposed to a variety of alcohol and xylene concentrations, the slices were sealed with neutral gum.

The IHC staining for eIF4E and cyclin D1 proteins in ovarian cancer samples was carried out with ready-to-use EnVision™+ Dual Link System-HRP methods (Dako, USA). Briefly, each section was regularly dewaxed through xylene followed by rehydration with a gradient of alcohols; subsequently, high-temperature antigen retrieval was achieved by heating the sections for 15 min in a 10 mM citrate buffer (pH 6.0) in a microwave, and then the sections were immersed into 3% hydrogen peroxide (H₂O₂) in methanol for 15 min. Sections were incubated with 5% normal goat serum for 30 min at room temperature. After that, the sections were incubated with a primary antibody, including eIF4E (pS473) protein (Epitomics, Inc., USA) in 1 : 200 dilution and cyclin D1 protein (Cell Signaling, USA) in 1 : 200 dilution at 4°C overnight, and washed thoroughly with PBS, and a secondary antibody (Maixin Biotechnology Inc., China) conjugated with a labeled polymer-HRP was incubated at room temperature for 50 minutes. The color reaction was developed by using DAB (Maixin Biotechnology Inc., China). Positive and negative controls were included in every experiment.
Immunohistochemical staining was estimated independently by two pathologists. The eIF4E and cyclin D1 staining was scored as negative (<10% staining) and positive (>10% staining), respectively [19 ].