Axio observer 1 microscope

Primary alveolar macrophages and MLE cells were seeded on coverslips and then incubated with DiD-labeled RAW MVs for 1 hour. In separate experiments, to visualize DiD-labeled MVs via confocal microscopy, MVs were placed on poly-L-ornithine coated coverslips to encourage adherence for 6 hours. Thereafter both MVs and cells were washed, fixed, permeabilized with 0.5% triton-X 100 and incubated with 3% bovine serum albumin (Sigma-Aldrich) for 30mins. Slides were then incubated with 5µg/ml T1 alpha (ab109059; Abcam, Cambridge UK) or 5µg/ml CD45 (ab23910; Abcam) overnight in the dark at 4°C, followed by washing and incubation with secondary antibodies (1:1000) for 1 hour. After washing, slides were treated with 4’,6-diamidino-2-phenylindole (DAPI) intra-nuclear stain (1:10000) solution (pre-made) for 10mins. Coverslips were placed on slides with Mounting PermaFluor (ThermoFisher Scientific) and viewed using a Zeiss LSM880 NLO multiphoton confocal imaging system with Axio Observer 1 microscope. The objective lens used was a Plan Apochromat 40x/1.3 oil DIC UVVIS-IR. The imaging medium was oil and the temperature -20°C. The fluorochromes used were Alexa-Fluor 488 and Alexa-Fluor 594. CZI Images were acquired using Zen software, which were then exported as 16 bit Tiff images. No image processing software was used.

Hematoxylin and eosin staining was performed for histopathological evaluation of the tissues. After sacrificing a mouse, the imaging window was opened by removing a plastic c-clip and gently lifting the coverslip. In case of tumor experiments, the entire tissue including ovary, oviduct, fat pad and part of uterine horn was removed. Tissue was gently pulled up with a forceps, detached from the tissue-supporting petals of the imaging window and cut off with a scissors, approximately in the center of the uterine horn. As an endpoint of hormonal stimulation experiment, only the ovary was removed. In this case, the forceps were softly latched in between the ovary and the oviduct and pulled up to detach the ovary. Removed tissues were attached to small cardboards and fixated with 4% formaldehyde. 24 h later, they were transferred to 1% formaldehyde in DDW. Tissues were dehydrated and paraffinized with tissue processor TP 1050 (Leica, Germany), and embedded in paraffin blocks with EG 1160 (Leica, Germany). 4 μm-thick sections were taken from the ovarian cortex and the medulla. After placing the sections on the slides, they were dried and stained with an TST-40 automated stainer (Medite, Germany). After sealing with the coverslips the slides were imaged with the Panoramic MIDI automatic slide scanner (3D Histech, Hungary), or alternatively with Axio Observer.1 microscope (Carl Zeiss).