Target retrieval solution s1700

Tissues were fixed in 10% neutral buffered formalin and embedded in paraffin. Kidney sections were stained with Masson’s trichrome, and Sirius red [4 (link),47 (link)]. For immunohistochemistry of F4/80, antigen retrieval from deparaffinized sections was performed using Target Retrieval Solution S1700 (Dako, Carpinteria, CA, USA) in an autoclave at 120 °C for five min. An anti-F4/80 monoclonal antibody (1:100 dilution, MCA497, Bio-Rad, Hercules, CA, USA) was used as the primary antibody. For the quantitative analysis, the remaining tubules in the areas of the cortex were evaluated in Masson’s trichrome-stained sections, using ImageJ software (National Institutes of Health, Bethesda, MD, USA).

Lung biopsy specimens were obtained from autopsy cases, in which the patient died of severe pneumonia, and resected lung from a patient with early lung cancer was also examined. Tissues were fixed in 10% formalin and embedded in paraffin. Deparaffinized sections (5-μm-thick) were immersed in epitope retrieval solution (Target Retrieval Solution S1700; Dako North America, Inc., Carpinteria, CA, USA) and preheated at 120 °C for 10 min. After blocking endogenous peroxidase activity with 3% H₂O₂ for 15 min, slides were incubated overnight with a mouse anti-human CD206 monoclonal antibody (15 µg/ml; R&D Systems, Minneapolis, MN, USA) or IgG2b at 4 °C. Subsequently, sections were incubated with visualization reagent (ChemMate Envision kit; Dako Japan, Inc., Tokyo, Japan) for 30 min, followed by counterstaining with hematoxylin.

Lung and pleural specimens were obtained from one patient with early lung cancer and PTB by partial resection and three patients with TB pleurisy by using thoracoscopy under local anaesthesia. Tissues were fixed in 10% formalin and embedded in paraffin. Deparaffinized sections (5-μm thick) were immersed in epitope retrieval solution (Target Retrieval Solution S1700; Dako North America, Inc., Carpinteria, CA, USA) and preheated at 120 °C for 10 min. After blocking endogenous peroxidase activity with 3% H₂O₂ for 15 min, slides were incubated overnight with a mouse anti-human CD206 monoclonal antibody (15 µg/ml; R&D Systems, Minneapolis, MN, USA) or IgG2b at 4 °C. Subsequently, sections were incubated with visualization reagent (ChemMate Envision kit; Dako Japan, Inc., Tokyo, Japan) for 30 min, followed by counterstaining with haematoxylin.

Mice were perfused with 4% paraformaldehyde in 0.1% phosphate buffer and postfixed for 24 h in the same fixative. The head tissue including the OE and the OB was decalcified in 10% EDTA solution, pH 7.0, and then embedded in paraffin. Coronal sections were cut (4 μm thick) and mounted on silane-coated slides. For antigen retrieval, deparaffinized sections were autoclaved at 121 °C for 20 min in Target Retrieval Solution (S1700; Dako). Immunohistochemistry was performed using an anti-NQO1 (rabbit polyclonal, 1:300; Cell Signaling Technology) antibody. Immunoreactivity was detected by incubation for 1 h at room temperature with a donkey antirabbit Alexa Fluor 594 (1:100; Invitrogen) antibody.
Serial coronal sections of the OB were prepared; for each section, the DI domain (NQO1-positive region) and the DII domain (NQO1-negative region) were distinguished by the anti-NQO1 antibody [19 (link)]. The DI and DII domains were then reconstructed (in three dimensions) from the coronal section to determine the DI and DII domains in the axial section (control sample). The number of responding glomeruli within the DI and DII domains was counted. Glomeruli with an immunostaining intensity exceeding two SDs of the mean background intensity for connective tissue under the lamina propria were considered NQO1-positive.

Some mice were intracardially perfused with 4% paraformaldehyde in 0.1 M phosphate buffer and decapitated, and the head was post-fixed for 24 h in the same fixative. The nasal tissues, including the OE, were decalcified using 10% EDTA solution, pH 7.0, and embedded in paraffin. Coronal sections (4 μm thick) were cut and mounted on silane-coated slides. Deparaffinized sections were autoclaved for 10 min in Target Retrieval Solution (S1700; Dako) for antigen retrieval. Immunohistochemistry was performed using anti-OMP (goat polyclonal, 1:4,000 dilution; Wako Chemicals). The immunoreaction was detected using the Histofine Simple Stain MAX-PO secondary antibody system (Nichirei) for goat anti-OMP.

IHC and IF studies using ASCL1 (clone 24B72D11.1, catalog number 556604, BD Biosciences, San Jose, CA) and NEUROD1 (clone EPR17084, catalog number ab205300, Abcam, Cambridge, MA) specific antibodies were carried out on archival formalin-fixed paraffin-embedded tissues. In brief, 5 μm paraffin sections were de-waxed and rehydrated following standard protocols. Antigen retrieval consisted of steaming for 40 min in Target Retrieval Solution (S1700, Agilent, Santa Clara, CA). Slides were then washed and equilibrated in TBS-Tween buffer (Sigma, St. Louis, MO) for 10 min. Primary antibodies were applied at a dilution of 1:25 at 37 °C for 60 min. For chromogenic studies, immunocomplexes were visualized by applying secondary detection reagents of the UltraVision™ Quanto Detection System (catalog number TL-060-QHD, Thermo Fisher, Waltham, MA) following the manufacturer’s instructions. Sequential dual-IF labeling studies were carried out using Tyramide SuperBoost kits (Thermo Fisher, Waltham, MA). All bright-field slides were imaged using a Ventana DP200 system (Roche Diagnostics, Indianapolis, IN). Fluorescence images were acquired on a Cytation 5 Cell Imager (Biotek, Winooski, VT). All the slides have been evaluated by an expert pathologist and the stainings have been replicated a minimum of three times.