G block

Antibodies and reagents used for histological experiments are as follows: Primary antibodies are: KIM-1 (MAB1817, R&D systems), AIM (rab2 rabbit polyclonal for IHC of mouse and human kidney specimens); #11 and 12 (for human free AIM ELISA) established in our laboratory, partly purchasable from Transgenic Inc.), S100A9 (AF2065, R&D systems, NE, USA). Secondary antibodies and related reagents are: G-Block (Genostaff, Tokyo, Japan) and HISTOFINE simple stain mouse MAX-PO (R, Rat, or G) (for nucleus; NICHIREI, Japan). Specimens were analysed using an inverted microscope: IX83 (Olympus) and a research slide scanner: SLIDEVIEW VS200 (Olympus).

Frozen sections were rinsed with 10 mM PBS at room temperature for 10 min, fixed with 4% PFA for 30 min at 37 °C, and washed again with distilled water. Then, sections were treated with 0.2% HCl for 10 min, followed by the reaction with 1 μg/ml proteinase K (Takara Bio, Shiga, Japan) for 10 min at 37 °C. After rinsing with PBS, sections were washed with G-Wash (Genostaff, Tokyo, Japan). Hybridization was performed in a humid chamber at 50 °C overnight using digoxigenin-labeled RNA probes, Col1a1 (NM_053304, nt3946-4887) diluted with G-Hybo (Genostaff). After the hybridization, the sections were washed with 50% formamide in G-Wash for at least 30 min at 50 °C and rinsed with TBST. Blocking was then performed with G-Block (Genostaff) for 15–30 min. After reaction with alkaline phosphatase (AP)-labeled anti-digoxigenin antibody (1:2000, Roche, Basel, Switzerland) at room temperature for 1 h, the sections were rinsed with TBST, followed by rinse with distilled water. The sections were reacted with BM Purple AP (Roche) as a substrate for 1 h at room temperature, after which they rinsed with PBS and were stained with Nuclear Fast Red solution as a counter staining. The sections were finally sealed with G-Mount (Genostaff). Periapical lesion and alveolar bone were observed under the optical microscope.

Colon tissues were fixed in 4% (w/v) paraformaldehyde (Wako) for 18 h at 4°C and embedded in paraffin blocks. Sections were stained using hematoxylin and eosin (H&E) and histological scores were assigned by a trained and blinded pathologist, as previously described (31 (link)). Eight pathological changes, including the extent of inflammatory cell infiltration, goblet cell reduction, decreased crypt density, crypt hyperplasia, thickening of the muscle layer, extent of submucosal tissue inflammation, crypt abscess, and ulceration, were rated on a 0–3 scale from normal to severe. The sum of each score (maximum 24) was used as the histological score. For immunofluorescence analyses, fixed tissues were incubated with 30% sucrose (Wako) in phosphate-buffered saline (PBS) for 18 h at 4°C and embedded in O.C.T. compound (Sakura Finetek, Osaka, Japan). After freezing at −80 °C, cryostat sections were blocked with G-Block (GenoStaff, Tokyo, Japan) for 15 min at room temperature. Samples were stained using Alexa Fluor™ 488-conjugated WGA overnight at 4°C. Nucleus staining was performed using 4′,6-diamidino-2-phenylindole (DAPI; Dojindo, Kumamoto, Japan). All images were obtained using a laser scanning confocal microscope (FV3000; Olympus).

Paraffin-embedded sections of lung tissues were de-paraffinized with xylene and rehydrated through an ethanol series and PBS. A microwave pretreatment was applied for antigen retrieval. Endogenous peroxidase was blocked with 0.3% H₂O₂ in methanol for 30 min, followed by incubation with G-Block (Genostaff) and the avidin/biotin blocking kit (Vector). The sections were incubated with anti-CD4, CD8, granzyme B, and perforin (Cell Signaling Technology) antibodies at 4 °C overnight. For the subsequent reaction, biotin-conjugated anti-rabbit Ig (DAKO) and peroxidase-conjugated streptavidin (Nichirei) were used. Peroxidase activity was visualized by diaminobenzidine. The sections were counterstained with Mayer’s hematoxylin.

Kidney sections for light microscopy analysis were fixed in 4% paraformaldehyde phosphate buffer. Sections were stained with periodic acid-Schiff. Glomeruli were digitally photographed, and the images were imported to the ImageJ software (National Institutes of Health, Bethesda, MD, USA; https://imagej.nih.gov/ij/) and analyzed morphometrically [20 (link)]. For immunohistochemistry, the tissue sections were deparaffined using xylene and rehydrated through an ethanol series and PBS. The antigen retrieval was performed by microwave treatment, with Citrate buffer, pH 6. Endogenous peroxidase was blocked with 0.3% H₂O₂ in methanol for 30 min, followed by incubation with the G-Block (Genostaff, Tokyo, Japan) and avidin/biotin blocking kit (Vector, CA, USA). The sections were incubated with an anti-CD31 rabbit monoclonal antibody (Cell Signaling, MA, USA) at 4°C overnight. They were incubated with biotin-conjugated anti-rabbit Ig (Dako, CA, USA), for 30 min at room temperature, followed by the addition of peroxidase-conjugated streptavidin (Nichirei, Tokyo, Japan) for 5 min. The peroxidase activity was visualized using diaminobenzidine. The sections were counterstained with Mayer's Hematoxylin (MUTO, Tokyo, Japan), dehydrated, and then mounted with Malinol (MUTO).

The samples were fixed with FAA solution (3.7% formaldehyde (methanol-free), 5% glacial acetic acid, 50% ethanol, pH 7.4). Tissue sections were deparaffined with xylene, and then rehydrated through an ethanol series and PBS. They were blocked with G-Block (Genostaff, Tokyo, Japan) for 10 min at room temperature and rinsed in TBS 3 times for 5 min at room temperature. Then, 2 µg/ml anti-BPG4 rabbit polyclonal antibody or normal rabbit IgG (Dako, Glostrup, Denmark, #X0936) was labeled at 4 °C overnight, and rinsed in TBS-T (TBS with 0.1% Tween20) twice for 5 min at room temperature followed by TBS. Donkey anti-rabbit IgG secondary antibody, Alexa Fluor™ Plus 647 (Invitrogen #A32795, 1:200 dilution) were added for 60 min at room temperature for detection of BPG4, and nuclei were counterstained with DAPI (Dojindo, Kumamoto, Japan). Tissue sections were rinsed in TBS 3 times for 5 min at room temperature and mounted with ProLong Gold (Invitrogen #P36934). Samples were observed with an ECLIPSE Ni microscope (Nikon, Tokyo, Japan). Experiments and observations were performed by Genostaff.

Rats were sacrificed on day 29 for immunohistochemical analyses. The ankles were decalcified and embedded in paraffin. After deparaffinization, the tissue sections were pre-treated with citrate buffer (pH 6.0) for 40 minutes at 80°C. Endogenous peroxidase activity was disrupted with 0.3% H₂O₂ in methanol. Nonspecific protein binding was blocked with G-Block (Genostaff) for 10 minutes. Endogenous biotin, biotin receptors, and avidin binding sites were blocked with the avidin/biotin blocking kit (SP-2001, Vector Laboratories). Mouse monoclonal anti-Hsp47 antibodies (Enzo Life Sciences) and mouse IgG2b (isotype control) were applied (2 μg/ml) over night at 4°C. Slides were washed in TBS-T and incubated with biotinylated goat anti-mouse antibodies (Dako). The signal was amplified with horseradish peroxidase-conjugated streptavidin (Nichirei Bioscience) and detected with 3,3′-diaminobenzidine. Cartilage destruction was scored from 0 (no cartilage loss) to 3 (complete loss of articular cartilage) [43 (link)]. The Hsp47-positive lining area was adjusted to the linear horizontal length (μm²/μm) of the analyzed lining [12 (link)].