Masson goldner trichrome staining kit

Veet hair removal cream was purchased from Reckitt Benckiser (France). White Vaseline was purchased from Korea-ione Co. Ltd. (Gyeonggi-Do, Korea). Silmazin 1 % cream was purchased from Dong-Wha Pharm. Co. Ltd. (Seoul, Korea). Rat interleukin-10 (IL-10) enzyme-linked immunosorbent assay (ELISA) kits and rat transforming growth factor beta 1 (TGF-β1) ELISA kits were purchased from Cusabio Biotech Co. Ltd. (Wuhan, Hubei Province, China). Rat tumor necrosis factor alpha (TNF-α) ELISA kits and rat vascular endothelial growth factor (VEGF) ELISA kits were purchased from Koma Biotech Inc. (Seoul, Korea). The Masson-Goldner trichrome staining kit was purchased from Merck in south Korea (Seoul, Korea), and hematoxylin, eosin Y alcoholic, and Acid Alcohol · Histo^TM were purchased from BBC Biochemical Co. (USA). Ammonium hydroxide ACS reagent was purchased from Sigma-Aldrich Co. Inc. (USA), and Harris ethyl alcohol and xylene were purchased from J.T.Baker^Ⓡ (Japan).
In the present study a rotary evaporator (Eyela Co., Japan), ELISA Plate Reader: VersaMax (Molecular Devices Co., USA), digital camera (Sony Corporation, Japan), micro high speed centrifuge (Vision Scientific Co. Ltd., Korea), HM440E microtome (Carl Zeiss, Germany), Olympus DP70 digital microscope camera and Olympus DP controller software (Olympus Imaging America Inc., USA) were used.

To fix the fat tissues removed from the rats, they were fixed in a 10% neutral formalin (Sigma‐Aldrich) solution for 24 h, and then the tissue samples were placed in tissue tracking cassettes and placed in a tissue tracking device ThermoScientific™ Citadel 2000 During the procedures on the device, the tissue was dehydrated by keeping it in a series of 50% (2 times), 60%, 70%, 80%, 96%, and 100% (2 times) ethyl alcohol (Merck GmbH). Afterwards, the tissues were clarified with the xylol (Merck GmbH) series, and then they were blocked using tissue embedding cassettes using metal base molds from the paraffin blocking device (Leica EG 1150 H). Sections of 4–5 μm thickness were taken from the obtained paraffin blocks using a Rotary microtome (Leica RM2255), and staining was carried out on a staining device (Leica ST5020) with the Masson‐Goldner trichrome staining kit (Merck GmbH).

Visualization of bone structure was done on 3 μm thick histological sections of decalcified bone following Masson-Goldner trichrome staining kit (Merck, Watford, UK), performed according to the manufacturers’ instructions. Tartrate resistant acid phosphatase (TRAP) staining of osteoclasts (3 μm) and identification of osteoblasts using morphological criteria on 3 μm thick histological sections were performed as previously described [37 (link)]. The number of osteoblasts (N.Ob/B.Pm), osteoblast surface (Ob.Pm/B.Pm), the number of osteoclasts (N.Oc/B.Pm), and the osteoclast surface (Oc.Pm/B.Pm) were determined on two non-serial sections using a Leica RMRB upright microscope with a 10× objective and OsteoMeasure software (Osteometrics, Decatur, GA, USA). Bone cell numbers per mm/trabecular bone was determined on all trabecular surfaces 125 μm away from the growth plate. All histomorphometric parameters were based on the report of the ASBMR Histomorphometry nomenclature and performed in a blinded fashion [38 (link)].