Harris hematoxylin

MPM tissue samples were fixed in 10% v/v buffered formalin and then paraffin-embedded. Tissue sections (4 µm-thick) were deparaffinized and rehydrated. The antigen unmasking technique was performed using EDTA-based Novocastra Epitope Retrieval Solutions (Leica Biosystems), pH 6, in a thermostatic bath at 98 °C for 30 min. The sections were then brought to room temperature and washed in PBS. After neutralization of the endogenous peroxidase with 3% v/v H₂O₂ and Fc blocking by a specific protein block (Novocastra, Leica Biosystems), the samples were incubated ON at 4 °C with the HAS1, HAS2, HAS3 and C1q primary antibodies. Staining was revealed using a polymer detection kit (Novocastra, Leica Biosystems) and the AEC (3-amino-9-ethylcarbazole, Dako, Denmark) or DAB (3,3′-diaminobenzidine) substrate chromogen. The slides were counterstained with Harris Hematoxylin (Novocastra, Leica Biosystems).

Ovarian tissues were fixed with 10% neutral buffered formalin (BBC, Washington, IL, USA) and embedded in paraffin. The paraffin block was serially cut into 4-μm ovary sections and these tissues were deparaffinized in a 60 °C dry oven and with xylene and ethanol. The deparaffinized tissues were then washed under tap water. The specimens were stained with Harris hematoxylin (Leica Biosystems, Wetzlar, Germany) for 7 min, and the specimens were dipped in 0.1% HCl for 2 s and counterstained with alcoholic eosin Y solution (Sigma–Aldrich). The stained slides were scanned for whole ovaries by 3D HISTECH (The Digital Pathology Company, Budapest, Hungary). The follicles were counted at 100-μm intervals in serially sectioned slides, and the total follicles were defined as primordial, primary, secondary, antral and atretic follicles, according to previous reports [39 (link)]. To analyze the number of follicles in ovarian tissue, the number of each follicle was counted.

For the immunohistochemical analysis, human normal and neoplastic tissues, including lung, breast, ovary, and stomach samples, were selected from the archives of the Department of Pathology, University of Palermo. Immunohistochemistry (IHC) was performed using a polymer detection method. Briefly, tissue samples were fixed in 10% v/v buffered formalin and then paraffin embedded. 4 µm-thick tissue sections were deparaffinized and rehydrated. The antigen unmasking technique was carried out using Novocastra Epitope Retrieval Solutions, pH 9 (Leica Biosystems) in a PT Link pre-treatment module (Dako) at 98°C for 30 min. Sections were then brought to room temperature and washed in PBS. After neutralization of the endogenous peroxidase with 3% v/v H₂O₂ and Fc blocking by a specific protein block (Novocastra, Leica Biosystems), samples were incubated overnight at 4°C with rabbit anti-human SP-D (dilution 1:300) polyclonal antibodies (MRC Immunochemistry Unit, Oxford, UK). Staining was revealed via polymer detection kit (Novocastra, Leica Biosystems) and AEC (3-amino-9-ethylcarbazole, Dako, Denmark) substrate-chromogen. Slides were counterstained with Harris Hematoxylin (Novocastra, Leica Biosystems). Sections were analyzed under the Axio Scope A1 optical microscope (Zeiss) and microphotographs were collected through the Axiocam 503 color digital camera (Zeiss) using the Zen2 software.

For histological analysis, Hematoxylin & Eosin (H&E) staining was performed as followed: sections were thawed at room temperature for 5 minutes, fixed in 2% paraformaldehyde for 15 minutes and subsequently washed in running tap water for 5 minutes. Tissue sections were then immersed in Harris Hematoxylin (Leica Biosystems) for 3 minutes, washed in warm running tap water for a further 3 minutes before differentiating in 1% acid alcohol. Sections were washed in warm running tap water for 3 minutes prior to immersing in Eosin (Leica Biosystems) for 40 seconds. Sections were washed briefly in water (10 dips) before dehydrating through a series of alcohol solutions from 70% to 100%, cleared through 4 changes of xylene and finally cover slipped using Permount mounting media. Digital images were captured using a TissueScope 4000 slide scanner (Huron Technologies).

Ovarian tissues were fixed with 10% neutral buffered formalin (BBC, Washington, USA), embedded in paraffin, and serially sectioned into 4 μm ovaries. Sectioned ovarian tissues were deparaffinized in a 60 °C dry oven and by xylene and ethanol. Deparaffinized tissues were washed under tap water. The slides were dipped in Harris hematoxylin (Leica Biosystems, Wetzlar, Germany) for 7 min, dipped in 0.1% HCl for 2 s and counterstained with alcoholic eosin Y solution (Sigma-Aldrich). The stained slides were scanned for whole ovaries by 3D HISTECH (The Digital Pathology Company, Budapest, Hungary). The follicles were counted every 100 μm in serially sectioned slides and were defined as the total follicles, including primordial, primary, secondary, and preovulatory follicles, and the antral follicles according to previous reports [18 (link)].