Automated tissue processor

Isolated aorta from each group was fixed in 10% formalin (v/v) for 24–48 h. The tissue was excised and processed using an automated tissue processor (Leica, Wetzlar, Germany) and subsequently embedded in Paraplast (Sigma-Aldrich, St. Louis, MO, USA). Afterward, the tissue was sectioned at 5 µm thickness using a microtome (Leica RM2235, Walldorf, Germany) before being stained with hematoxylin and eosin (H&E), and Verhoeff–Van Gieson’s (VVG) method, respectively [37 (link)]. H&E-stained aortic sections were used to measure the thicknesses of the aortic wall, tunica media, tunica adventitia (without the perivascular adipose tissue), and lumen diameter. Meanwhile, VVG staining was performed to compare the lining of internal lamella in the tunica media layer. For histomorphometry of the H&E-stained aorta, photographs were taken from five different non-overlapping fields (n = 10/group) using a 40 × objective. Each photograph was used for the morphometry analysis using the straight segmented or freehand lines tools of the cell sense system in Image analyzer (Olympus, Tokyo, Japan). The mean value of the five fields was used as a representative for each rat.

The dissected ovaries were fixed in 10% neutral buffered formalin (Sigma Aldrich) at room temperature overnight. The fixed ovaries were processed through serial ethanol, xylene, and paraffin baths in the automated tissue processor (Leica). The paraffin-embedded ovaries in blocks were cut serially into 6 µm sections, and every fifth section of the whole ovary was stained with H&E and analyzed for follicle counts. The follicles were categorized into primordial, primary, secondary, and antral follicles based on the follicle size as well as the shape and number of the surrounding layer(s) of granulosa cells (25 (link)). Only follicles with visible oocyte nuclei were counted. The total number of follicles counted for each category was then multiplied by a correction factor of 5 (the sampling rate) to be representative of the population of the entire ovary. The follicle counting was conducted independently by two experienced observers, and the average number was used for statistical analyses.

IP implants were kept intact within the peritoneum. The overlying skin was removed to isolate the entire peritoneal cavity, fixed for 48 hours in Bouin’s Solution, then dissected into three 1 cm sections before being fixed for another 24 hours. Bouin’s solution was rinsed out with successive rinses of 1xPBS before being placed in 70% ethanol for FFPE processing.
SQ implants were dissected from the underlying muscle with overlying skin attached to the implant. Samples were fixed overnight in 10% neutral buffered formalin (NBF) overnight, and then rinsed in distilled water before being placed in 70% ethanol for formalin-fixed paraffin-embedded (FFPE) processing.
VML implants were dissected and collected similarly to those previously described for flow cytometry. Samples were incubated in 10% NBF for 48 hours before rinsing in distilled water and placing in 70% ethanol for FFPE processing. All models were dehydrated in a graded ethanol series and cleared in xylenes before paraffin infiltration using an automated tissue processor (Leica). Samples were mounted on paraffin blocks, and 5 μm sections were taken before being baked for 3 hours at 60°C and stained with hematoxylin and eosin (H&E, Sigma) or Masson’s trichrome (Sigma) as per manufacturer’s instructions. Slides were imaged on an EVOS microscope.