Eclipse ti2 u microscope

CNE2-EBV cells carrying the Akata-EBV-GFP genome were induced by 20 ng/ml 12-O-tetradecanoylphobol 13-acetate (TPA; Beyotime) and 2.5 mM sodium butyrate (NaB; Sigma Aldrich) for 12 h. After 72 h in culture, the supernatant was collected, centrifuged and then filtrated through a 0.45 μm filter to remove cell debris. The resulting virus, named CNE2-EBV-GFP, was concentrated 100 × by centrifugation at 50,000 g for 2.5 h and re-suspended by RPMI 1640 without FBS. The CNE2-EBV-GFP virions were stored at − 80 °C. A NIKON Eclipse Ti2-U microscope was used to capture images of non-induced CNE2-EBV cells as well as induced cells at 72 h post induction.
Akata-EBV cells carrying Akata-EBV-GFP were resuspended in RPMI 1640 without FBS and induced by 0.8% (v/v) goat anti-human IgG (Tianfun Xinqu Zhenglong Biochem. Lab). The medium was changed after 6 h induction. The Akata-EBV-GFP virus collection procedures and storage were the same as those used for the CNE2-EBV-GFP virus. A NIKON Eclipse Ti2-U microscope was used to capture images of non-induced Akata-EBV cells as well as induced cells at 72 h post induction.

Aliquots of spermatozoa (1 × 10⁶) contained in the capacitation solution were mixed in a 1:1 ratio with 4% paraformaldehyde (PFA) in PBS for 20 min at room temperature, for fixation. The samples were then centrifuged at 2000× g for 5 min. They were washed twice with 0.05 M glycine in PBS and once with PBS. Then, a total of 0.05 × 10⁶ spermatozoa were air-dried on a microscope slide and stained with a Coomassie solution (0.22% Coomassie Blue G-250, 50% methanol, 10% acetic acid, and 40% water) for 2 min as described in [82 (link)]. The slides were thoroughly rinsed with distilled water, air-dried, and mounted with a coverslip and Eukitt mounting medium (EUK-NEO-100, Microptic, Barcelona, Spain). This staining method allows to distinguish acrosome-intact spermatozoa from acrosome-less spermatozoa (Figure S1). A minimum of 200 spermatozoa was analyzed per replicate for each condition using an inverted Eclipse Ti2-U microscope (Nikon, Amstelveen, The Netherlands).

Following immunostaining, brain sections were imaged with a Nikon Eclipse Ti2-U microscope equipped with Appo Fluor (× 4 NA 0.2, × 20 NA 0.75, × 40 NA 0.6) objectives at room temperature using air as the imaging medium and a Qi2 monochrome camera. Nikon Elements software was used to acquire images from a camera and to analyze the percent area for each staining. A researcher blind to the treatment of each mouse analyzed the total area of positive staining as a percentage of total image area (n = 3–4 sections/mouse; 5–7 mice per group).