Aminoethyl carbazole system

Section (6-µm thickness) from paraffin-embedded mouse skin and lungs were deparaffinized and incubated overnight at 4 °C with mouse mAbs to CD3 (1:200; Nichirei Bioscience), F4/80 (1:1600; Abcam), and α-SMA (1:200; DAKO), followed by incubation with peroxidase-labeled secondary antibody (Nichirei BioScience) and color development with the aminoethyl carbazole system (Nichirei BioScience) [28 (link)]. Immunostained cells were counted in three high-power microscopic fields. Each section was examined independently by two investigators (T.C. and N.O.) in a blinded manner.

Sections (6 μm in thickness) from paraffin-embedded mouse skin were incubated for 120 minutes at room temperature with monoclonal antibodies (mAbs) to CD3 (1:200; Nichirei Biosciences, Tokyo, Japan), F4/80 (1:1600; Abcam), and p-Smad3 (1:50; Santa Cruz Biotechnology), then with peroxidase-labeled secondary antibody (Nichirei Biosciences), followed by color development with the aminoethylcarbazole system (Nichirei Biosciences). CD3⁺ cells, F4/80⁺ cells, and p-Smad3-positive cells were counted under a high-power microscopic field (the Hall section for CD3⁺ cells and distinct fields for the F4/80⁺ cells and p-Smad3-positive cells). Each section was examined independently by two investigators (TC and NO) in a blinded manner.

Paraffin-embedded mouse skin sections (6 μm in thickness) were incubated for 120 min at room temperature with monoclonal antibodies (mAbs) to CD3 (1:200; Nichirei Bioscience), F4/80 (1:100; Abcam), α-SMA (1:200; Abcam), CD31 (1:200; Abcam), Snail1 (1:100, Gene Tex), Snail2 (1:100; Invitrogen), or cytokeratin 5 (1:500; BioLegend), then with HRP-labeled secondary antibody (Nichirei BioScience), followed by color development with the aminoethyl carbazole system (Nichirei BioScience). CD3⁺ cells and F4/80⁺ cells were counted under a high-power microscopic field (CD3⁺ cells and F4/80⁺ cells in three distinct fields). Each section was examined independently by two investigators (T.C. and N.O.) in a blinded manner. The area of the CD31-positive vessels, Snail1, or SNAIL2 was quantified as pixel counts in three distinct fields of each three skin sections using Adobe Photoshop Elements version 12.