Cx21 microscope

Retroperitoneal fats were fixed for 48 h in formalin-alcohol, inserted in paraffin, cut into 3-μm thick specimens and finally hematoxylin and eosin (HE) staining was done. Sections were examined by a CX21 microscope (Olympus, Tokyo, Japan) and images were captured at 20 magnification. Adipocyte space diameters were measured in two different microscopic fields and the average was then calculated.
Furthermore, samples were collected from ovarian tissues and fixation was done in 10% paraformaldehyde for 1 day. After that, sections were dipped in water followed by serial dilutions of alcohol. Sections were then inserted in paraffin wax and were sectioned at 4-μ. The cut tissue specimens were stained by HE or Masson's trichrome and inspected by a light microscope (39 ) to detect pathological changes. A calibrated digital microscope camera (Tucsen ISH1000, Yuscen Photonics Co. Ltd., China) using Olympus® CX21 microscope, with resolution of 10 megapixels (3,656 × 2,740 pixel for every image). All Masson's trichrome stained sections were captured at original magnification 400x (Objective 40x), UIS optical system (Universal Infinity System, Olympus, Tokyo, Japan).

HE, TUNEL and immunohistochemical (IHC) staining were carried out as reported by us previously [39 (link)]. HE staining was used to detect the pathological changes. Apoptotic cells in tumor tissues were stained with a TUNEL Apoptosis Detection Kit (Beyotime) according to the manufacturer’s protocol. For histological analysis, tumors were fixed overnight in 10% neutral buffered formalin, embedded in paraffin and sectioned at 5-μm thickness using a Leica RM2265 microtome. IHC staining was carried out with an EnVision Detection System HRP. A rabbit/mouse (DAB+) kit (Agilent) was used following the manufacturer’s instructions. Endogenous peroxidase was blocked by incubation with 0.3% hydrogen peroxide for 15 min. Antigen retrieval was performed by boiling the slides in citrate buffer (10 mM, pH 6.0) in a water bath for 20 min. After being rinsed and blocked with 5% bovine serum albumin (BSA), the slides were incubated overnight at 4 °C with primary antibodies, followed by 1 h with labeled Polymer-HRP at room temperature. Subsequently, the slides were exposed to DAB+ Chromogen. Counterstaining with hematoxylin was carried out. After mounting, the slides were observed under an Olympus CX21 microscope, scanned with a high-resolution digital slide scanner (Pannoramic 250, 3DHistech), and quantified by ImageJ software.

H&E and IHC staining were performed as previously reported^{46 (link)} with a small modification. H&E staining was used to detect pathological changes. For histological analysis, formalin-fixed, paraffin-embedded tumors were sectioned, and slides were deparaffinized using xylenes (Thermo Fisher Scientific Inc.). Endogenous peroxidases were quenched with 3% hydrogen peroxide in methanol. Staining was performed using antibodies against BRCA1 (1: 100), BRCA2 (1: 100), RAD51 (1: 50), γ-H2AX (1: 50), P-gp (1: 200), BCRP (1: 200), PAR (1: 250), or Ki67 (1: 500). Counterstaining was performed using Mayer’s hematoxylin (Dako, Glostrup, Denmark). Images were observed under an Olympus Cx21 microscope, scanned with a high-resolution digital slide scanner (Pannoramic 250, 3DHistech), and quantified using ImageJ software.