Cellsens dimensions 2

Specimens were stained for 3 min in hematoxylin (Gill No. 3, Sigma-Aldrich, Munich, Germany), washed 2 times with H₂O followed by differentiating in acidic alcohol (1% HCl in ethanol) for 3 s and bluing under running tap water for 10 min. Counterstaining was conducted with eosin (Eosin Y, Sigma-Aldrich, Munich, Germany) for 10 s. Dehydration was performed in increasing alcohol series and Roti-Histol^®. Finally, sections were mounted with Entellan^®. One area per animal was evaluated based on the region of trauma. Six animals were used for each group based on the power analysis conducted for the animal experiment application (license number: 1183). Pictures were taken with the UC30 color camera at X10 and X40 magnification. All stainings were acquired with an Olympus IX81 and analyzed with the Olympus software cellSens Dimensions 2.3 (Build 18987).

Paraffin-embedded, 5 μm thick cross-sections of skeletal muscle and lung tissue were dried for 24 h at 40°C with subsequent deparaffinization in Roti-Histol^® and hydration in decreasing alcohol series before having been used for Ki67 staining. The samples were boiled for 15 min at 450 W in a microwave using citrate demasking solution (14746, CST). After a cooling period and several washes with water as well as PBS the samples were stained overnight at 4°C with the Ki67 rabbit mAb (12202, CST) at a dilution of 1:250. The sections were washed, and the secondary staining was performed with one drop Histofin Simple Stain Max Po Anti-Rabbit (414142F, medac diagnostika). Sections were incubated for 20 min. The samples were washed and a counterstaining with hemalaun (109249, Sigma-Aldrich) was performed. Finally, sections were mounted with Entellan^®. Two areas per animal were evaluated based on trauma region. Four animals were evaluated from each group and a picture was taken with the UC30 color camera at X10 and X40 magnification. All stainings were acquired with an Olympus IX81 and analyzed with the Olympus software cellSens Dimensions 2.3 (Build 18987). Percentage was calculated as Ki67 positive cells normalized to the total amount of cells in the field.

The deparaffinized lung sections were stained using SR solution (0.1% in aqueous saturated picric acid) for 1 h and differentiated in 0.5% acetic acid. Subsequently, the slides were counterstained with hematoxylin for 10 min to stain the cell nuclei as described by Xu et al. [32 (link)]. Increasing alcohol series and Roti-Histol® was used for dehydration of the sections that were then covered with Entellan®. One area per animal out of a total of 6 mice per group was analyzed for the evaluation of the trauma. This number is based on the power analysis conducted for the animal experiment application (license number: 1183). Pictures were taken with the UC30 color camera at X10 and X40 magnification. All stainings were acquired with an Olympus IX81 microscope and analyzed with the Olympus software cellSens Dimensions 2.3 (Build 18,987).