Mayer s hemalaun solution

For Trap staining, sections were fixed with 4% PFA, titrated to pH5 using a buffer containing Natriumacetat (Merck, Darmstadt, Germany) and Natriumtartrat-dehydrat (Merck, Darmstadt, Germany), and washed with distilled water. Thereafter, callus sections were stained in the presence of Naphtol AS-MX- Phosphate, Fast Red Violet LB Salt, and N,N-Dimethylformamid. A counterstain was carried out with Mayer’s Hemalaun solution (Merck, Darmstadt, Germany). Osteoclasts were identified as TRAP-positive cells with ≥ 3 nuclei and adherent to the bone surface. Osteoblasts were identified as mononuclear cells adherent to the bone surface with typical cuboidal morphology. Static and cellular histomorphometric parameters were assessed according to the guidelines of the American Society for Bone and Mineral Research [19 (link)] using ImageJ software.

Endothelial cell basal medium and pericyte growth medium were purchased from PromoCell (Heidelberg, Germany). Dulbecco’s modified Eagle’s medium (DMEM) low glucose was from Lonza (Cologne, Germany). Human TNF-α was from Provitro (Berlin, Germany). MA was purchased from Santa Cruz Inc. (Heidelberg, Germany). Fluorescein isothiocyanate-labeled dextran 150,000, rhodamine 6 G and peroxidase-labeled-streptavidin were purchased from Sigma-Aldrich (Munich, Germany). Mayer’s hemalaun solution was from Merck (Darmstadt, Germany). 3-Amino-9-ethylcarbazole was obtained from Abcam (Cambridge, UK). Ketamine (Ursotamin) was from Pharmacia GmbH (Erlangen, Germany) and xylazine (Rompun) was from Bayer (Leverkusen, Germany).

For characterization of NK cells in spleen and liver, 14 μm thick cryostat sections were cut and placed on glass slides. Every third section was selected for immunohistochemical analyses performed with the alkaline phosphatase antialkaline phosphatase technique as previously described [15 (link)]. The primary mouse anti-rat antibody directed against the NK-RP1 receptor (CD 161, clone 10/78, AbD Serotec, Puchheim, Germany, 1 : 1000) was used. The sections were stained with Fast Blue (spleen) or Fast Red (liver; Fast Blue/Red TR Salt; Sigma-Aldrich), counterstained with hematoxylin (Mayer's hemalaun solution; Merck, Darmstadt, Germany) for 15 s, and mounted in glycergel (Dako Cytomation, Glostrup, Denmark). For the quantitative analysis in the liver and spleen of six animals per group, five sections and eight visual fields per section were examined for each animal (resulting in 38,4 m² investigated area/group/organ). In the spleen sections, four visual fields in the red pulp and four fields in the transition zone between white and red pulp were selected. The quantitative immunohistological analyses were performed blinded to the treatment conditions using the Image J software (US National Institutes of Health, Bethesda, MD, USA).

After deparaffinization and rehydration in xylene and alcohol solutions with descending concentrations, slides were stained in Mayer’s hemalaun solution (Merck, Darmstadt, Germany) for 3 min, shortly differentiated in 0.1% hydrochloric acid and washed under running tap water for 10 min. Afterwards, slides were stained with a 0.5% eosin solution (Eosin G, Carl Roth, Karlsruhe, Germany), shortly washed in Aqua dest. and differentiated using ascending concentrations of alcohol.

For TIRC7 immunohistochemical staining of CCA TMAs, rabbit polyclonal anti-TIRC7 antibody (CellAct, Dortmund, Germany) was used [20 (link),22 (link)]. In brief, 5 µm sections were cut from the formalin-fixed paraffin-embedded samples and deparaffinized and rehydrated according to standard protocols. Sections were then subjected to epitope retrieval at pH8 prior to incubation with anti-TIRC7 antibodies (1:500) for 30 min at room temperature. For detection, the LSAB system was used (Dako REAL Detection System, Agilent Technologies, Santa Clara, CA, USA). For FOXP3 immunohistochemistry, epitope retrieval was performed at pH6. After blocking of endogenous peroxidase (Dako REAL Peroxidase Blocking Solution, Agilent Technologies) sections were incubated with anti-FOXP3 (clone PCH101, 1:50, Thermo Fisher Scientific, Waltham, MA, USA) for 30 min at room temperature followed by incubation with secondary antibody (rabbit anti-rat, 1:1000, Dianova, Hamburg, Germany) for 30 min at room temperature. For detection, EnVision+ Single Reagent (Agilent Technologies, Santa Clara, CA, USA) was used. Nuclei were stained using Mayer’s hemalaun solution (Merck Millipore, Burlington, MA, USA) and sections coverslipped with Kaiser’s glycerol gelatin (Carl Roth GmbH, Karlsruhe, Germany).