Bx43 microscope

The ability of neutrophils to move from the mucosal region of the endometrium through the epithelium in response to the external environment was assessed as follows:
Endometrial samples (~1 cm²), which included the surface epithelium, were harvested from three animals for each group (heifers exposed to LF semen, heifer exposed to HF, and CTRL heifers—no semen exposure) within 30 min of slaughter and fixed in 10% neutral buffered formalin for 48 h. Following processing into paraffin, tissue blocks were sectioned at 5 μm thickness using a Leica microtome and stained with hematoxylin and eosin. The sections were examined using an Olympus BX43 microscope fitted with an image analyser (Image—Pro Premium; Media Cybernetics, MD, USA). Using a 63x oil immersion objective, 20 and 40 fields of view (FOV) were selected per sample. Neutrophils within the columnar epithelium region only were quantified. The count was expressed as the average number of neutrophils per FOV per group. On average, 106 FOV were analyzed per treatment group.

HCC samples were fixed overnight with 4% buffered formalin before embedding in paraffin. Paraffin sections were cut at a thickness of 5 µm and dried for 24 h. After dewaxing and rehydration, sections were boiled in citrate buffer for 30 min for antigen retrieval. Then, sections were cooled in citrate buffer, rehydrated with PBS and incubated with 3% H₂O₂ for 10 min to block endogenous peroxidase activity. Nonspecific binding sites were blocked with 10% bovine serum albumin (BSA) for 30 min. Sections were stained with the ZCCHC13 antibody (1:100) at 4 °C overnight. Meanwhile, sections incubated with IgG were used as negative controls. After the incubation, sections were washed three times with PBS and incubated with a horseradish peroxidase-conjugated secondary antibody (Jackson ImmunoResearch) for 1 h at room temperature. Peroxidase activity was visualized with a 0.05% solution of the chromogen 3,3′-diaminobenzidine (Sigma) before counterstaining with a haematoxylin solution. Finally, slides were observed under an Olympus BX43 microscope and analyzed with Image-Pro Plus 6.0 software (Media Cybernetics).

Uterine biopsies from three heifers from each treatment were used for immunohistochemical localization IL-1 alpha. The formalin-fixed paraffin-embedded endometrial biopsies were sectioned at 5 μm thickness and stained using primary antibody Interleukin-1 alpha (#P420A, Thermo Fisher Scientific). Samples underwent antigen retrieval in 0.01 M citrate buffer and were blocked for endogenous peroxidases using 0.3% H₂O₂. A horseradish-peroxidase-conjugated goat anti-rabbit IgG H&L secondary antibody (Abcam, Cambridge, UK) was applied and developed with 3,3’diaminobenzidine substrate (DAB substrate, Abcam) before being mounted. Representative pictures were taken using an Olympus BX43 microscope fitted with an image analyser (Image—Pro Premium; Media Cybernetics). A negative control with no primary antibody was applied.