Tsa plus fluorescence kit

Paraffin-embedded synovial tissues of RA were sectioned, deparaffinized, and antigen-retrieved with 0.01 M citric acid of pH 6.0. The clinical background of the patients is shown in Supplementary Table 3. Immunohistochemistry was performed with Sox4 antibody (HPA029901, rabbit polyclonal, Sigma Aldrich, 1:100) and detected with EnVisionTM Detection System (DAKO). ELS formation (Score E) and the presence of Sox4-positive cells within the infiltrating cell population without ELSs (Score SN) or with ELSs (Score SE) were assessed by a semi-quantitative four-point scale (none, 0; mild, 1; moderate, 2; and severe, 3). The total Sox4 expression score corresponds with the sum of the SN and SE scores. Triple immunofluorescence staining was performed with CXCL13 antibody (BCA1, rabbit polyclonal, Thermo Fisher, 1:100), Sox4 (HPA029901, rabbit polyclonal, Sigma Aldrich, 1:30), CD3 (Clone PS1, mouse monoclonal, Leica Microsystems, 1:200), CD4 (Clone 1F6, mouse monoclonal, Leica Microsystems, 1:100), CXCR5 (Clone 51505, mouse monoclonal, R&D Systems, 1:1000), and PD-1 (Clone NAT105, mouse monoclonal, Abcam, 1:50) and detected with TSA Plus Fluorescence Kit (PerkinElmer, Inc.). Fluorescence imaging analysis was performed using the FSX100 Fluorescence Microscope (Olympus).

Analyzed specimens comprised of FFPE tissues from IgG4-RD patients retrieved from the archive of the department of Diagnostic Pathology, Kyoto University Hospital: pancreas (n = 5), bile duct (n = 3), retroperitoneum (n = 1), aorta (n = 1), kidney (n = 2), salivary gland (n = 6), lung (n = 4), ureter (n = 1), and lymph node (n = 6) from 29 patients between January 2007 and August 2015. Paraffin embedded sections from IgG4-RD patients were immunostained with the following antibodies: alpha-smooth muscle actin (1A4, Sigma Aldrich, St. Louis, MO, USA, 1 : 300), CD3 (F7.2.38, DakoCytomation, Glostrup, Denmark, 1 : 50), CD11c (5D11, Cell marque, Rocklin, CA, 1 : 50), CD123 (6H6, eBioscience, San Diego, CA, USA, 1 : 100), CD20 (Clone L26, mouse monoclonal, DakoCytomation, 1 : 1), CD68 (PGM1, DakoCytomation, 1 : 10), galectin-3 (1 : 100), IgG (EPR4421, Abcam, 1 : 50), and IgG4 (HP6025, Nichirei Biosciences Inc., Tokyo, Japan, 1 : 1). The REAL™ EnVision™/HRP detection system (DakoCytomation, Glostrup, Denmark) was used to detect immunohistochemical signals. Double immunofluorescence staining was performed using the TSA Plus Fluorescence kit (PerkinElmer Inc., Waltham, MA, USA).

Whole-mount in situ hybridization (WISH) was performed as previously described [11 (link)], using the following probes: csrp3, nkx2.5, hand2, tp53, fosl2, piezo1, ilk. Antisense riboprobes were synthesized in vitro using T7 RNA polymerase (NEB) with DIG RNA Labeling Mix. Transcription templates were amplified from cDNA using specific primers listed in Supplementary Table 1. The signal was detected using anti-digoxigenin-AP (Roche) and visualized by NBT/BCIP substrate (Roche). Images were taken by a Nikon SMZ18 stereo microscope. Fluorescence in situ hybridization (FISH) of csrp3 expression on cryosections was performed as previously described [17 (link)], signals were detected using anti-digoxigenin-POD (Roche) and visualized by TSA plus fluorescence kit (PerkinElmer), subsequently conducted immunofluorescence with Tnnt2 antibody as described above. FISH images were obtained using a Leica SP8 confocal microscope.

Liver tissues were fixed in 10% formalin and embedded in paraffin. Slides with 5-µm tissue sections were deparaffinized in xylene and rehydrated in ethanol. Antigen retrieval was performed in citrate buffer (pH 6.0) using microwave treatment. Primary antibodies for HBcAg (HBV core protein, Dako) (1:800), IL-32 (1:100), and/or β-gal were incubated for 1 h in a humidified chamber at room temperature, followed by detection with secondary antibody (1:50) at room temperature by staining with a TSA Plus fluorescence kit (Perkin Elmer, Waltham, MA, USA). The slide was then placed in citrate buffer (pH 6.0) and heated using a microwave oven. The images were acquired with a fluorescence microscope at ×100 magnification.