Mayer s hemalaun

For histochemistry and immune‐histochemistry, paraffin‐embedded skin samples were cut into 4 μm sections. Haematoxylin and eosin (HE) staining using Mayer's Hemalaun (1.09249.2500, Merck) and Eosin Y (1.15935.0100, Merck) was done in a linear slide stainer (Leica ST4040). For Masson–Goldner trichrome staining, kits were used according to the manufacturer's recommendations (12043, 14604, Morhisto). Automatic scanning of full slides in 40x magnification was done using the VS‐120‐L Olympus slide scanner 100‐W system and processed using the Olympus VS‐ASW‐L100 program. Evaluation of the epidermal thickness and murine vessel quantification was done according to published work (Ebner‐Peking et al., 2021 (link)), both measured in the Olympus VS‐ASM‐L100 program. Per group, four biological and at least three technical replicates were included.

Slides were subjected to BrdU-staining for evaluation of hepatocytes proliferation. The staining procedure was based on a modified protocol of Sigma Inc. After deparaffinization and rehydration (Xylene 30 minutes, 100% ethanol 3 minutes, 90% ethanol 3 minutes, 70% ethanol 3 minutes, distilled water 3 minutes, TBS for 5 minutes), tissue sections were treated with prewarmed 0.1% trypsin solution (Sigma, St. Louis, MO) at for 40 minutes, followed by denaturation of the DNA with 2 N HCl (Merck, Darmstadt, Germany) at for 30 minutes. In the following step, sections were incubated with 1∶50 monoclonal anti-BrdU antibody (Dako, Hamburg, Germany) at for 1 hour, followed by an alkaline-phosphatase labeled secondary anti-mouse antibody (Power Vision, Immunovision Technologies, USA) for 1 hour at room temperature. Color reaction was performed using the Fast Red Substrate System (sensitive) (Dako, Hamburg, Germany) for 10 minutes. The sections were counterstained with Mayers hemalaun (Merck, Darmstadt, Germany) for 10 seconds, and cover slipped using ImmuMount (Shandon, Pittsburgh, PA). Acquisition of the digital images was performed using the same equipment as the one used during HE staining after BrdU-staining procedures.

Collagenase NB 4G was purchased from SERVA Elektrophoresis GmbH (Heidelberg, Germany). Neutral red, fluorescein isothiocyanate (FITC)-dextran 150,000, rhodamine 6G and Hoechst 33,342 were purchased from Sigma-Aldrich (Taufkirchen, Germany). Mayer’s hemalaun was purchased from Merck (Darmstadt, Germany), ketamine (Ursotamin®) from Serumwerke Bernburg (Bernburg, Germany) and xylazine (Rompun®) from Bayer (Leverkusen, Germany). HepatoQuick® and EPO-β (recombinant human EPO, NeoRecormon®) were purchased from Roche (Basel, Switzerland). The antibody anti-EPOR (BS1424R) was purchased from Fisher Scientific (Hampton, USA). The antibody anti-CD31 (DIA310) was purchased from Dianova (Germany). The antibody anti-green fluorescent protein (GFP) was purchased from Rockland Immunochemical Inc. (Limerick, USA) and the antibodies anti-insulin as well as 3-amino-9-ethylcarbazole (AEC Substrate System) from Abcam (Cambridge, UK).