Blocking one histo

Cells seeded on collagen coating glass-bottom culture dishes (Matsunami Glass Ind., Ltd.) were transfected with 250 nM mPCS1f or BRO (mPCS1f/PNA(C8)) and incubated for 48 h at 37 °C and 5% CO₂. After rinsing thrice with 1× PBS, cells were fixed at room temperature with 4% formaldehyde for 10 min and permeabilized with 0.1% NP-40 in 1× PBS for 10 min. Fixed cells were blocked in Blocking One Histo (Nacalai Tesque) at room temperature for 30 min and then incubated in 20× diluted Blocking One Histo (Nacalai Tesque) with primary antibodies at room temperature for 2 h. After washing thrice with PBS-T (5 min per wash), cells were incubated in 20× diluted Blocking One Histo with secondary antibodies at room temperature for 1 h. Finally, cells were washed thrice with PBS-T (5 min per wash) and nuclei were stained with Hoechst 33258 (Dojindo) for 10 min. The cells were mounted using Fluoromount (Diagnostic Biosystems). Confocal images were generated using confocal laser scanning microscopy (LSM710 microscope, Carl Zeiss Co., Ltd.) and analyzed by ZEN software 3.5. 093. 00001 (blue edition). The 3D structure was rendered in PyMol (Version 2.4.1).

Twenty-four h after the last FAP inhalation, the trachea was cannulated with a 22-G needle attached to a syringe, and 4% paraformaldehyde was injected. Then the lungs were harvested and fixed in 4% paraformaldehyde at 4 °C overnight. The fixed tissues were embedded in paraffin and sliced into 4-µm sections. The sections were incubated with Blocking One Histo (0634904; Nacalai, Japan). After blocking, they were incubated with goat anti-mouse IL-33 polyclonal Ab (AF3626; Abcam plc, UK) in a humidified staining box at 4 °C overnight. They were then incubated with 0.3% hydrogen peroxide solution and 0.1% sodium azide for 20 minutes at room temperature to block endogenous peroxidase activities. Then the sections were incubated with horseradish peroxidase-conjugated secondary Ab (N-Histofine Simple Stain MAX PO Goat; 414351; Nichirei Biosciences Inc., Japan) for 30 minutes at room temperature. After washing, IL-33-producing cells in the lung sections were detected with an HRP/diaminobenzidine (DAB) system. Nuclei were counterstained with Mayer’s hematoxylin for 1 minute. The sections were mounted using Malinol (20092; Muto Pure Chemicals, Japan) and imaged using a BZ-X710 (KEYENCE, Japan).

LPS-primed peritoneal macrophages were seeded on coverslips, stimulated with nigericin (1 μM) for 30 min and then fixed and permeabilized in 2.5% formaldehyde and 0.5% Triton X-100. The cells were incubated with anti-ASC pAb followed by Alexa Fluor 488-conjugated anti-rabbit IgG pAb. Nuclei were counterstained with Hoechst 33342. The cells were examined under a fluorescence microscope (Keyence). Coronal slices from ischaemic brain were embedded in Tissue-Tec O.C.T. Compound (Sakura Finetechnical Co., Ltd) and flash frozen in liquid nitrogen and stored at −80 °C. Sections of 8 μm in thickness were fixed for 15 s with acetone, then rinsed with PBS. Blocking with Blocking One Histo (Nacalai Tesque) was applied for 1 h at room temperature. Section were washed with PBS and then incubated with anti-BTK pAb, anti-F4/80 mAb, anti-NeuN mAb, anti-MAP2 mAb or anti-NLRP3 mAb followed by Alexa Fluor 488- or 546-conjugated goat anti-rabbit, mouse or rat IgG (H+L) or incubated with FLICA.

Indirect immunofluorescent antibody test (IFAT) using tick section was performed as described previously34 (link). Briefly, ticks were fixed overnight in a 4% paraformaldehyde phosphate buffer solution (pH 7.4) that included 0.1% glutaraldehyde and was embedded in paraffin. Cut sections were fixed on glass slides and deparaffinized in xylene. The sections were rehydrated with a graded series of alcohol and PBS, followed by trypsin treatment. They were then blocked using Blocking One Histo (Nacalai Tesque, Kyoto, Japan). They were then incubated for 1 hour at 37 °C with mouse anti-BoP29 serum (1:100) diluted by Can Get Signal Immunostain Immunoreaction Enhancer Solution (Toyobo). Sections treated with pre-immune mouse sera (1:100) were used as a control. After washing, sections were reacted with Alexa Fluor 488 goat anti-mouse IgG as a secondary antibody (1:1000) and mounted with VECTASHIELD Mounting Medium with DAPI (Vector Laboratories, Burlingame, CA, USA). The slides were examined under a confocal laser scanning microscope (LSM 710, Carl Zeiss, Oberkochen, Germany) with LSM Software ZEN 2012 (Carl Zeiss). For the IFAT with egg squashed smear, randomly selected eggs of 10 days post oviposition were used and treated as described previously32 (link).

Ly-6G⁺ cells treated with α-toxin and G-CSF were cytospinned onto microscopic glass slides and blocked with Blocking One Histo (Nacalai Tesque, Inc., Kyoto, Japan). The samples were then incubated with a primary antibody against G-CSFR. After washing with PBS, samples were incubated with the secondary antibody conjugated with Alexa Fluor 488.
Femoral muscles were embedded in OCT compound (Sakura Finetek Japan, Tokyo, Japan), and cryosectioning of the frozen tissue was performed using a cryostat microtome (Leica, IL, USA). Sections were blocked with Blocking One Histo and incubated with primary antibodies. Finally, the sections were incubated for 1 h with Alexa Fluor 546 goat anti-rabbit IgG and Alexa Fluor 488 goat anti-rat IgG. The antibodies were diluted in DAKO Antibody Diluent (DAKO, Glostrup, Denmark). Nuclei were stained with 4’,6-diamino-2-phenylindole. Images were captured on a confocal laser-scanning fluorescence microscope (Nikon A1, Nikon instruments, Tokyo, Japan).

The excised tibialis anterior (TA) muscles were snap-frozen in liquid nitrogen-cooled isopentane, cut into 10-μm-thick sections, and stained with hematoxylin and eosin (H&E) or for succinate dehydrogenase (SDH) activity. Immunofluorescence staining of muscle cryosection was performed after blocking with Blocking One Histo (nacalai tesque, Kyoto, Japan). The sections were incubated with primary antibodies diluted in PBS at 4°C overnight. After washing with PBS, sections were incubated with secondary antibodies labeled with Alexa 488 or Alexa 568 for 1 h at room temperature and mounted using Fluro KEEPER Antifade Reagent (nacalai tesque). The antibodies used for immunofluorescence staining are shown in Table 1. Liver sections were immersed in 4% paraformaldehyde, embedded in paraffin, cut into 5-μm-thick sections, and stained with H&E or Sirius Red. The liver pathologic grade was determined by a specialist using the SAF score (Bedossa et al., 2012 (link)) by assessing steatosis, activity (inflammation), and fibrosis. An all-in-one fluorescence microscope (BZ-X800, Keyence, Osaka, Japan) was used for imaging. Muscle fiber cross-sectional area (CSA), the percentage of type 1 fibers, and the percentages of the sections that were Sirius red-positive were calculated using BZ-H4C analysis software (Keyence).