Hematoxylin gill s formula

TAAR1 expression was obtained in tissue micro arrays (TMAs), which were created from a collective of formalin-fixed, paraffin-embedded ovarian cancer samples. The experiment was initiated by deparaffinization of the slides (4 µm) by xylol. In order to inactivate endogenous peroxidase, 3% H₂O₂ in methanol (20 min) was used. Rehydration of the slides was obtained by a descending ethanol gradient. A pressure cooker filled with Epitope Retrieval Solution pH 9.0 (Novocastra by Leica, Wetzlar, Germany) provided the preparation of the tissue for heat induced epitope retrieval. Next, blocking solution was applied to prevent non-specific binding of the primary antibodies. The slides were incubated at room temperature for 1h with anti-TAAR1 (polyclonal rabbit IgG, Atlas Antibodies by BIOZOL, Eching, Germany) diluted 1:100. Antibody reactivity was detected using ImmPRESS Anti-Rabbit IgG Polymer Kit (Vector Lab. by BIOZOL, Eching, Germany) according to the manufacturer’s protocol, followed by application of substrate and chromogen (3,3′-diaminobenzidine Agilent Technologies, Waldbronn, Germany). In the next step, the slides were counterstained with hematoxylin Gill’s formula (Vector Lab.) and cover slipped.

The second immunohistochemical staining followed a different protocol, as it was performed in the Institute of Pathology of the University Hospital, LMU Munich. Instead of PBS, TRIS buffer (pH = 7.5) was used for rinsing. The dewaxing was followed by heat pretreatment with Target Retrieval Solution (Agilent Technologies, Santa Clara, CA, USA). After antigen retrieval, the endogenous peroxidase was blocked with 7.5% aqueous hydrogen peroxide. The 20 min incubation with blocking serum (ImmPRESS Reagent Kit Anti-Rabbit IgG; Vector, Burlingame, USA) was followed by a 60 min incubation at RT with the primary antibody (CCL22/MDC, No 500-P107, 1:200; PeproTech, Rock Hill, USA). The sections were then incubated for 30 min with anti-rabbit Ig (ImmPRESS Reagent Kit Anti-Rabbit IgG; Vector Laboratories, Burlingame, CA, USA). Visualization with DAB+ for three minutes and counterstaining with Hematoxylin Gill’s Formula (Vector Laboratories, Burlingame, CA, USA) followed. Aquatex (Merck, Darmstadt, Germany) was used for covering.

Available tissue microarrays (TMA) of primary EwS tumors containing 2 cores of each sample, with a diameter of 1 mm, as well as internal controls were stained for CALCB. Analysis were carried out with approval from LMU Munich ethics committee.
For IHC, 4-μm sections were cut and antigen retrieval was carried out by heat treatment using target unmasking fluid (PanPath, Budel, Netherlands). Slides were incubated for 60 min at room temperature with a rabbit polyclonal anti-CALCB antibody (bs-0791R, Bioss Antibodies Inc., MA, USA; dilution 1:120). Then slides were incubated with a secondary anti-rabbit IgG antibody (Vectastain ABC-Kit Elite Universal, Vector laboratories, Burlingame, CA, USA) followed by target detection using DAB plus (Agilent Technologies, Santa Clara, CA, USA). Counterstaining was performed with Hematoxylin Gill’s Formula (Vector). Intensity of CALCB staining was scored independently by two researchers on a scale from 0 to 2 (0 = majority of the cells is negative for CALCB staining, 1 = majority of the cells shows moderate CALCB staining, and 2 = majority of the cells shows strong CALCB staining). Specificity of the anti-CALCB-antibody was assured by determination of immunoreactivity scores (IRSs) using the Remmele and Stegner scoring system^{35 (link)} in 6–11 representative high-power fields (×40) per xenograft with/without qRT-PCR-confirmed knockdown of CALCB.