Blocking solution

Mouse brains were perfusion-fixed with 4.0% paraformaldehyde (PFA) 4 weeks after cranial window surgery over the primary visual cortex (Fig. 1e, f, Supplementary Fig. 2a, b). Brains were removed, fixed overnight in 4% PFA, and cut into 50-μm thick coronal sections using a vibratome (7000smz; Campden Instruments, Leicestershire, UK). Slices were then incubated in 10% blocking solution (NACALAI TESQUE, INC., Japan) containing 0.2% Triton X-100 for 30 minutes at room temperature, followed by incubation with a primary antibody against glial fibrillary acidic protein (GFAP, 1:1,000, FUJIFILM Wako Pure Chemical Corporation, Japan) overnight (~16 h) at 4°C. After washing with 0.1% Tween 20 in PBS, slices were incubated with secondary antibody (1:500; Alexa Fluor 594 donkey anti-mouse IgG; Thermo Fisher Scientific, Waltham, MA, USA) for 4 h at room temperature. Nuclei were counterstained with 1:2000 Hoechst 33258 (FUJIFILM Wako Pure Chemical, Japan). Finally, stained slices were examined using a confocal microscope (Leica TCS SP8 STED 3X FALCON, Leica, USA) with an HC PL APO CS2 ×10/0.40 NA dry objective lens. Mean Hoechst 33258 and GFAP fluorescence emission levels were measured at 15 ROIs from three mice in each condition using ImageJ and normalized per 300 × 300 µm².

To confirm the location of APP770-V5 and FNDC5-HA, we examined immunofluorescence staining of SH-SY5Y cells transiently expressing these molecules. We washed prepared cells by PBS and fixed them by 4% paraformaldehyde for 15 min at room temperature. Then, these cells were permeabilized by 0.1% Triron-X and blocked by using Blocking solution (Nacalai Tesque, Japan). We used the mouse monoclonal anti-V5 antibody (1:1000; Sigma) and the rabbit polyclonal anti-HA antibody (1:1000; Sigma) for the primary antibodies to detect APP and FNDC5, and then labeled them by Alexa Fluor 594-conjugated goat anti-mouse (1:2000; Life Technologies, MA, USA) and Alexa Fluor 488-conjugated mouse anti-rabbit (1:2000; Life Technologies), respectively. As the mounting agent, we used NucBlue Fixed Cell Stain ReadyProbes reagent from Life Technologies. These cells were observed using a laser confocal scanning microscope (FV10i-LIV, Olympus, Japan).

Manually, the cells were fixed in 4% paraformaldehyde for 5 min at 25°C. Thereafter, the cells were washed twice with Tris-buffered saline containing 0.2% Tween-20 (TBS-T) and treated with a blocking solution (Nacalai Tesque) for 30 min at 25°C. The first antibody-containing blocking agent was added to the cells and incubated overnight at 4°C with paraffin sealing to prevent evaporation. The cells were washed thrice with TBS-T and immersed in the second antibody-containing blocking agent for 1 h at room temperature. After three washes, the fluorescent signals were observed using a fluorescence microscope (Nikon Instruments, Tokyo, Japan). The primary and secondary antibodies used are listed in Supplementary Table S1.

Testes were immersed in OTC embedding compound (Tissue-Tek, Sakura, Tokyo, Japan) and were frozen at −20 °C. Sections (10 μm thick) were prepared using a cryomicrotome (HM 500 OM; Microm, Walldorf, Germany). Mature sperm were removed from the cauda epididymis and spotted onto glass slides (Matsunami Glass, Osaka, Japan). Samples were fixed with 80% methanol (for detection of KPNA6 and C1QBP) or 4% PFA (for detection of CENPJ) at 4 °C for 20 min before being permeabilized in PBS containing 0.01% Triton X-100 for 10 min. Each sample was treated with a blocking kit (Vector Labs, Newark, CA, USA) and blocking solution (Nacalai Tesque, Kyoto, Japan). The samples were incubated at 4 °C for 16 h with each antibody. After washing with TBS-T, the sections were incubated at room temperature for 90 min with biotinylated anti-rabbit Igs antibody (Amersham Bioscience). After washing with TBS-T, signals were observed using streptavidin/avidin-conjugated Alexa Fluor 568 (Amersham Bioscience). The sections were counterstained with 4′,6-diamidino-2-phenylindole (DAPI; Nacalai Tesque) and/or 20 µg/mL FITC-conjugated peanut aglutinin (FITC-PNA) (Sigma, St. Louis, MO, USA), washed, and examined under a fluorescence microscope. TRA54 and 98 were used to identify the chromatoid body and the nuclei of germ cells, respectively [17 (link),69 (link)].

Paraffin-embedded ALI-cultured HBECs were sectioned at a thickness of 5 µm. Sections were depleted of paraffin, autoclaved at 120 °C for 10 min in citrate buffer (pH 6.0) for antigen retrieval, and exposed to a blocking solution (#03649-64; Nacalai Tesque, Kyoto, Japan) for 10 min before incubation overnight in a humidified chamber at 4 °C with primary antibodies or isotype control. Immune complexes were detected by incubation with secondary antibodies for 1 h at room temperature, after which the sections were mounted in mounting medium containing DAPI (#H-1200; Vector Laboratories, Newark, CA). Images were acquired with a BZ-X810 fluorescence microscope (Keyence, Osaka, Japan) and analyzed with Image J software.

C. merolae logarithmic growth cells (10 ml at OD₇₅₀ = 0.5) were collected and fixed with a cold fixation buffer (Ohnuma et al., 2008 (link)) and incubated at −30°C for 5 min. Fixed cells were then collected and resuspended in methanol and stored at −30°C until use. The cells were washed with 1 × PBS and blocked with 5% v/v, blocking one solution (hereafter, blocking solution, Nacalai Tesque, Inc., Kyoto, Japan) for 30 min at room temperature. The cells were washed with blocking solution and incubated with an anti-FLAG antibody (Wako, Osaka, Japan, a dilution rate: 1/100) in blocking solution at 4°C overnight. The cells were washed with blocking solution and incubated with an Alexa Fluor 488 donkey antimouse antibody (Thermo Fisher Scientific, Waltham, MA, United States, a dilution rate: 1/100) in blocking solution at room temperature for 1 h while avoiding light. The cells were then washed with blocking solution, and the fluorescence signal was captured by fluorescence microscopy (BX-51, Olympus, Tokyo, Japan). Data were treated by Adobe Photoshop 2021 to enhance the contrast and light intensity of the fluorescence from the sample cells.

Western blotting analysis was performed as described with minor modifications [22 (link)]. Homogenate from the liver was prepared with a RIPA lysis buffer (150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 50 mM Tri-HCl (pH 7.4) supplemented with HaltTM Protease and Phosphatase Inhibitor Cocktail (Thermo Scientific, Tokyo, Japan). The same concentration of protein (20–40 µg per each sample) was resolved by SDS-polyacrylamide gel electrophoresis and then transferred to PVDF membranes. The membrane was blocked with a blocking solution (Nacalai Tesque), followed by incubation with the following primary antibodies: anti-FAS (diluted 1:1000; #3180; Cell Signaling Technology, CST), anti-ACC (diluted 1:1000; #3676; CST), anti-SCD-1 (diluted 1:1000; #2794; CST), anti-TNFα (diluted 1:800; #11948; CST), and anti-β-actin (diluted 1:3000; #4980; CST). After washing, each band was incubated with an HRP-conjugated anti-rabbit IgG secondary antibody (#7074; CST) and detected with the ECL Prime Western Blotting Detection System (GE Healthcare, Uppsala, Sweden). We captured each band images using the ChemiDoc XRS Plus imaging system (Bio-Rad, Hercules, CA, USA) and quantified the protein levels by analyzing the band intensities using ImageJ (NIH, Bethesda, MD, USA).