Nis elements f2.30 image acquisition software

The obtained spleen tissue was fixed in 4% paraformaldehyde at 4 ℃ for 48 h and embedded in paraffin. The paraffinised sections (0.3 μm) were dewaxed with water, and the antigens were heat-recovered at a high temperature. The sections were then blocked with H₂O₂ for 40 min at room temperature, washed with PBS, and then blocked with serum (Elabscience) for 40 min at room temperature. Paraffin sections were incubated overnight at 4 °C with antibodies against HLA-DR (BOSTER, CA, USA) and Caspase-3 (Cell Signaling Technology, Boston, MA, USA). The sections were then washed thoroughly with PBS and then incubated with the corresponding biotin-labelled secondary antibodies (Elabscience) for 20 min at room temperature at 37 °C. Thereafter, the sections were incubated with diaminobenzidine (DAB, Elabscience), developed, and counterstained with haematoxylin. The negative control was incubated with PBS instead of the secondary antibody. Brown-yellow or brown-yellow particles with a ring-shaped distribution indicate positive results. Images were captured using an NIS-Elements F2.30 image acquisition software (Laboratory Imaging, Hostivař, Czech Republic). The average optical densities (ODs) of the positive areas were measured with ImagePro Plus software (Media Cybernetics, Rockville, MD, USA), reflecting the expression level of the corresponding products.

5 × 10⁴DC2.4 cellswere plated on 24-well plate with chamber slides. The cells were washed twice with PBS, fixed with 4% paraformaldehyde (4 °C, 30 min), washed with PBS, and then treated with pre-cooled 70% ethanol (− 20 °C, 4 h). The cells were then washed with PBS, 100 µL of TUNEL equilibration buffer was added, and the cells were incubated for 5 min. The buffer was discarded, the excess liquid was blotted off with filter paper, and 50 µL TUNEL reaction solution was added. The samples were then covered with buffer, and then incubated at 37 °C for 60 min. The reaction solution was removed and the cells were washed with PBS for 5 min. The cells were then permeabilised using 0.1% Triton X-100 (containing 5 mg/mL BSA) in PBS, washing thrice for 5 min each time in PBS. DAPI counterstaining. Dropped onto adhesive slides and covered with cell slides. Finally, images were captured using NIS-Elements F2.30 image acquisition software (Laboratory Imaging). Statistical analyses were performed using ImagePro Plus software (Media Cybernetics).