Tp 1050

Animals were sacrificed by cervical dislocation while still being anaesthetised after the last imaging session. The C ring and the cover glass were removed from the imaging window, and the tissue was gently detached from the supporting petals. The desired amount of tissue, corresponding to the accessible field of view in the imaging window, was dissected with scissors and attached to a cardboard in the same orientation as visible on the images, and placed in 4% PFA. Twenty-four hours later, the tissues were transferred to 1% PFA. Tissue dehydration and paraffinisation were performed with tissue processor TP 1050 (Leica, Germany), followed by embedment in paraffin with EG 1160 (Leica, Germany). In all, 4-µm sections were cut approximately from the middle of the tumour, so the ovary and fat pad would also be visible. The slides were automatically stained with TST-40 (Medite, Germany). The imaging was performed with Panoramic MIDI slide scanner (3D Histech, Hungary).

Tumour tissues were preserved in 4% paraformaldehyde (PFA) immediately after resection from mice. They were then run through a series of automated processing steps executed in a Leica TP 1050 tissue processor. The resulting paraffin-embedded blocks were sectioned using American Optical microtome into 4 μm thick tissue sections and placed on slides. Sections were re-hydrated, antigen retrieval was performed in 0.01 M citrate buffer (pH 6), followed by blocking with PBS containing 5% serum and staining with primary antibodies (see Supplementary informatioin for details). Mounting solution, VectaShield® HardSet™ with DAPI, was used to seal the slides with coverslip.

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).

Samples for histology were fixed overnight in 4% paraformaldehyde (PFA) in PBS before being dehydrated through an ethanol gradient using a Leica TP 1050 vacuum tissue processor. Samples were embedded in paraffin and sectioned at 5 μm thickness using a microtome. Hematoxylin and eosin (H&E) staining was performed using an automated system (Tissue-Tek DRS, Sakura). Immunohistochemistry was performed by The Queen Mary University of London Barts Cancer Institute Pathology Service using the Ventana DabMap Horseradish Peroxidase Kit. Stained slides were scanned using a Nanozoomer Whole Slide Imager (Hamamatsu Photonics) to create virtual slides using NDP.View2 software. For cell type quantification, images of sections that contained areas of intact epithelium with at least 300 cells were used (overall 1–6 slides were assessed per donor). Images were reviewed in Fiji software and positively stained cells counted using the cell count function. In total, 12,568 (TP63), 9,651 (MUC5AC), 16,144 (FOXJ1) and 17,459 (CD45) cells were assessed for expression of these proteins.

After the fattening period animals from each group were slaughtered, and samples of the lungs, liver, kidneys, and jejunum were taken. These samples were fixed for 24 h in 10% neutral buffered formalin and absolute alcohol, and then passed through increasing concentrations of alcohol solutions and xylene in a tissue processor (Leica TP-1050) and embedded in paraffin blocks. Histological Sects. 4 µm thick, prepared using a sledge microtome (Leica SR-200) and stained with haematoxylin and eosin, were used for morphological analysis under a light microscope. For the liver samples, histochemical staining was additionally performed for the presence of neutral fats. For these analyses, the sections were fixed in 10% neutral buffered formalin, sliced in a freezing microtome (Cryotome FSE, Thermo Scientific), and stained with Sudan IV according to Daddi [17 ]. For visualization of glycogen, microscope sections of the liver were fixed in absolute alcohol and stained by the PAS method according to McManus [18 ].

Adult zebrafish were fixed in 4% PFA for 2 days, followed by decalcification in 20% EDTA for 1 week. The animals were cut at the mid-trunk level and processed in a Leica TP1050 tissue processor in preparation for paraffin embedding. The embedding station used was a Leica EG 1150H. Sagittal sections of the samples were cut at a thickness of 7 μm on a microtome (Thermo Fisher Scientific, microm HM325). Sections were arranged and fixed on Superfrost Plus^TM slides (Thermo Fisher Scientific) and then subjected to hematoxylin and eosin (H&E) staining to evaluate the gross morphology of the thyroid and count thyroid follicles. For each group, three zebrafish were analyzed. All sections with follicles were used to determine the number of thyroid follicles.

Twenty-four hours after the last administration of the study materials, the animals were euthanized and decapitated. The brains were carefully and rapidly excised, wet weight taken and then fixed in 4% paraformaldehyde in phosphate-buffered saline. The fixed brain samples were preserved for frontal cortex histological, histochemical and immunohistochemical analysis according to Bancroft and Gamble (2008 ) method. The frontal cortex (FC) was carefully dissected from the whole brain according to stereotaxic atlas guideline from Paxinos and Watson (2007 ) brain atlas. The tissues were processed using an automated tissue processor (LEICA TP 1050). The paraffin section was cut at 5 μm thickness and stained with Haematoxylin and Eosin (H and E) stain, Cresyl Fast Violet (CFV) stain and Glial Fibrillary Acidic Protein (GFAP) immunohistochemistry according to methods of Bancroft and Gamble (2008 ) and Akinrinade et al. (2015a ).