Mayer s hemalum solution

Consecutive sections stained for hematoxylin and eosin were used to classify the ovarian cancer and peritoneal tissue. Therefore, the embedded ovaries were serially sectioned, and tissue sections (3 μm) were placed onto SuperFrost Plus slides (VWR, Leuven, Belgium). Tissue sections were dewaxed in xylene, rehydrated in descending concentrations of ethanol, washed in distilled water, and stained with hematoxylin (Mayer’s hemalum solution, Merck, Darmstadt, Germany) for 3 min, followed by a rinsing with hydrochloric acid 0.1 % and by a wash in water and acetic alcohol before staining with eosin (Eosin Y-solution 0.5 % aqueous, Merck, Darmstadt, Germany) for 3 min. After dehydrating in ascending concentrations of ethanol and xylene, sections were mounted in Eukitt (Kindler, Freiburg, Germany).
The final tumor staging (TNM classification) was obtained from the report of the institutional pathologist and documented for each ovarian cancer patient for later analysis. Data on the intraoperative resection status of each tumor patient were obtained from the operation report.

Frozen liver sections (7-µm) were fixed with 5% formalin, washed, and oxidized with 1% (w/v) Periodic acid for 15 min. After washing, the slides were placed in Schiff’s reagent (Merck, Darmstadt, Germany) for 30 min as described [67 (link)], and were counterstained by Mayer’s Hemalum solution (Merck). For quantitative analysis, the percentage of PAS⁺ cells per high-power field (×200) to the total number of hepatocytes was determined using five random microscopic fields per animal.

HSC-2 cell lines were cultured for 4 days in CM of CD163⁺, CD204⁺, and CD206⁺ cells and used for invasion assays by using Corning^® BioCoat™ Matrigel^® Invasion Chambers (Corning Incorporated, Corning, NY, USA) As follows the product’s guidelines, three Matrigel and three control inserts were prepared for a 24 well culture dish. The CM (750 μl) of CD163⁺, CD204⁺, and CD206⁺ TAM was used as a chemoattractant in each well subsequently and the insert was filled with 500 μl of DMEM (Life Technologies Corporation) without serum and HSC-2 cell line (2.5 × 10⁴ cells/insert). The cells were cultured for 22 hours in a humid chamber with 5% CO₂ atmospheric state at 37 °C temp. Then the inserts were removed from the wells and Matrigel was cleaned off. After that cells were fixed with 100% methanol (Junsei Chemical Co.,Ltd., Tokyo, Japan) and then stained with Mayer’s hemalum solution (Merck KGaA, Darmstadt, Germany, 1:4 dilution) and Tissue-Tek eosin (Sakura Finetek Japan Co., Ltd, Tokyo, Japan) (Supplementary Method 1).
The numbers of cells on the membrane were counted in 4 mm² sections from five different high-power microscopic fields (400×; 0.0625 μm²). The average number of cells in the Matrigel insert membrane was divided with the average number of cells in the control insert membrane, and then multiplied with 100 to calculate the invasion percentage.