3 3 diaminobenzidine solution

The left femur was fixed in 10% NBF for 2 days, demineralized using 10% EDTA-2Na for 3 weeks, and then dehydrated with ethanol, clarified with xylene, and embedded with paraffin. Paraffin embedded tissue was sectioned on a rotary microtome. Endogenous peroxidase was blocked in 3% H₂O₂/Mt-OH for 15 min at room temperature. Then, 20 μg/ml proteinase K (Thermo fisher, PA, USA) was used for epitope retrieval for 10 min at 37 °C, for blocking, normal serum (Gibco, Gaithersburg, MD, USA) was reacted at room temperature for 30 min. The tissues were incubated with primary antibody diluted in 0.5% BSA, including anti-NFATc1 (1:100, Santa Cruz Biotechnology, CA, USA), anti-c-fos (1:100, Santa Cruz Biotechnology, CA, USA) and anti-cathepsin K (1:100, Santa Cruz Biotechnology, CA, USA), at 4 °C overnight. The tissues were then stored in 1:100 biotinylated secondary antibody (Vector Labs, Burlingame, CA, USA) for 60 min at room temperature. The tissues were incubated in horseradish peroxidase-streptavidin using ABC kit (Vector Labs, Burlingame, CA, USA) for 30 min at room temperature and stained with 3,3′-Diaminobenzidine solution (Vector Labs). The tissues were counterstained with hematoxylin, dehydrated and mounted. The immunohistochemical stained tissues were observed with a light microscope (100, 200×).

IHC for SCD was performed in lung adenocarcinoma tissue microarray (US Biomax, Rockville, MD, USA) according to the manufacturer’s instructions [37 (link)]. The prepared tissues microarray slides were incubated with SCD antibody (Cell-Signaling technology) for overnight and incubated with horse anti-rabbit secondary antibody (Vector Laboratories, Burlingame, CA, USA) for 2 h. Following treatment with 3,3’-diaminobenzidine solution (Vector Laboratories), slides were counterstained using hematoxylin, dehydrated in alcohol, and mounted in a xylene-based solution. The mean IHC staining intensity for SCD was quantified using ImageJ software and scored 0–3 as follows [38 (link)]: 0, no staining; 1, < 15 mean intensity; 2, 15–50 mean intensity; and 3, > 50 mean intensity.

Immunohistochemistry analysis was performed in five µm-thick frozen or formalin-fixed paraffin-embedded liver tissue sections using antibodies against CYP3A (Biotrend, Cologne, Germany), CYP1A, CYP2C (a gift from Dr. R. Wolf, Biochemical Research Centre, University of Dundee, Dundee, UK), CYP2E1, Arginase1 (Sigma-Aldrich Corp., St. Louis, MO, USA), GS (BD Bioscience, Heidelberg, Germany), and CPS1 (Abcam, Cambridge, UK) (Table 1). The following horseradish peroxidase-conjugated secondary antibodies were used: anti-rabbit IgG (Agilent, Santa Clara, CA, USA), anti-mouse IgG (Sigma-Aldrich Corp., St. Louis, MO, USA), and anti-rat IgG (Linaris GmbH, Heidelberg, Germany) (Table 1). In order to visualize the target signal, the tissues were stained with either 3,3′-diaminobenzidine solution (Vector Laboratories, Peterborough, UK) or AEC+ high sensitivity substrate chromogen (Agilent, Santa Clara, CA, USA). The nuclei were visualized by counter-staining with Mayer’s haematoxylin.

Expression of RasGRP4 protein levels was measured using immunohistochemistry (IHC). Paraffin sections (5 μm) of human DLBCL tissues and benign tissues were incubated at 60 °C for 15 min, deparaffinized with xylene, and rehydrated by graded alcohol. The slides were then incubated with 3% hydrogen peroxide and 0.2% Triton X-100. Antigen retrieval was performed at 95 °C for 30 min (the sections were placed in 0.01 M citrate buffer [pH 6.0]), then washed with PBS 3 times, followed by incubation with normal goat serum (Jackson Laboratory, 005–000-121, 1:200, final volume required for full coverage of specimens was based on the size of the sample) for 2 h. After that, the slides were incubated overnight with rabbit anti-human RasGRP4 antibody (Abcam, ab96293, 1:200) at 4 °C. On the next day, the slides were incubated with a biotinylated goat anti-rabbit IgG antibody (Jackson Laboratory, 111–035-003, 1:200, 30 min) and visualized with a streptavidin–biotin–peroxidase reaction (Vector Laboratories, 1:1, 30 min). After colorimetric reaction with 3,3′-diaminobenzidine solution (Vector Laboratories, 1:50, 3 min), the slides were dehydrated and mounted. Representative images were obtained with a Nikon ECLIPSE Ni microscope with color camera, and the expression of RasGRP4 protein was determined by measuring the average optical density (AOD) using Image-Pro Plus 6.0 software.