Ab955

Tissue blocks were sectioned at 4 µm on a graded slide and standard immunohistochemical techniques were carried out as previously described.⁵¹ For fluorescent immunocytochemistry, primary mouse monoclonal anti‐CD68 antibody (ab955, 1:100; Abcam, Cambridge, UK) and the primary rabbit polyclonal anti‐CD209 antibody (PA5‐78968, 1:1000; Thermo Fisher) were used and immunoreactivity was visualized after incubation with antirabbit secondary antibody coupled with Alexa Fluor‐568 or antimouse secondary antibody coupled with Alexa Fluor‐488 (Molecular Probes, Invitrogen). Slides were cover slipped with VECTASHIELD Mounting Medium for Fluorescence and imaged with a Nikon Eclipse TE2000‐U using NIS Elements Basic Research software (version 4.51). For standard immunocytochemistry, the same mouse monoclonal antibody for CD68 described above and the primary rabbit monoclonal anti‐CD3 antibody (ab16669, 1:100; ABCAM) were used. Immunoreactivity was visualized by incubation with chromogen diaminobenzidine for 5 min. Tissue sections were counterstained with hematoxylin, dehydrated through graded ethanol and xylene and coverslipped. Images were captured with an Olympus BX41 light microscope using SPOT Software version 5.1 (SPOT Imaging Solutions, Detroit, MI, USA).

Immunohistochemistry was used to investigate intracerebral IgG and IgM distribution. Briefly, paraffin wax-embedded tissues from humans or macaques were dewaxed in xylene and hydrated through graded alcohols. Endogenous peroxidase activity was suppressed by 3% H₂O₂ in PBS. Subsequently, sections were incubated with goat anti-IgG (anti-human IgG (gamma chain), 1/100, SAB3701291, Sigma-Aldrich, Saint-Louis, MI, USA), with rabbit anti-IgM (anti-human IgM, mu chain, 1/250, A0425), with polyclonal rabbit anti-Human C1q (1/100, A0136, Dako, Les Ulis, France), with anti-MBP (1/100, SMI94R BioLegend, San Diego, CA, USA), with rabbit anti-IBA1 (1/100, ab178846, Abcam, Cambridge, UK), or with mouse anti-CD68 (1/150, ab 955, KP1, Abcam). Tissues were then incubated with secondary antibodies coupled to fluorochromes Alexa 488 or Alexa 594 (ThermoFisher, Villebon-sur-Yvette, France). Alternatively, biotinylated rabbit-anti-goat, rabbit-anti-mouse, or goat-anti-rabbit antibodies were used as secondary antibodies, for 30 min at room temperature, followed by the avidin-biotin-peroxidase complex (Vectastain Elite ABC Kit, Vector Laboratories, PK 6100; Burlingame, CA, USA). Positive antigen-antibody reactions were visualized by incubation with 3,3-diaminobenzidine-tetrahydrochloride (DAB)–H₂O₂ in 0.1 M imidazole, pH 7.1 for 5 min, followed by slight counterstaining with hematoxylin.

We collected specimens from a local cohort of 21 BLCA patients to analyze the expression level of EPHB6 and its correlation with TME feature. These patients were classified into three groups: NMIBC-low grade (n=7), NMIBC-high grade (n=7), and MIBC-high grade (n=7). Tissue samples (4-µm-thick sections) were subjected to immunohistochemical staining for EPHB6, CD8, CD68 using EPHB6 (ab217542, 1:100, Abcam) CD8 (ab217344, 1:100 Abcam) and CD68 (ab955, 1:50, Abcam) antibodies, respectively. The immunohistochemical staining method was performed as described previously by Sun et al. (44 (link)). The staining score for EPHB6 was classified as high (staining in more than 50% of malignant cells), low (staining in less than 25% of malignant cells), or medium (staining in 25%-50% of malignant cells). The staining of CD8 and CD68 was only evaluated in intratumoral infiltrating leukocytes. The score was calculated independently by two pathologists from five high magnification (200) visual fields using CaseViewer2.2 (Thermo Fisher).

Geniposide (≥98% purity, as detected by high-performance liquid chromatography analysis) was purchased from Shanghai Winherb Medical Co. (Shanghai, China). TGF-β (T7039) was obtained from Sigma-Aldrich (St. Louis, MO, United States). ISO was purchased from Sigma-Aldrich Co. EX-527 was purchased from MedchemExpress (Sollentuna, Sweden). Antibodies against the following proteins were purchased from Abcam: TGF-β1 (ab66043), SIRT1 (ab110304), gp91 (ab80508), 4-hydroxynonenal (4HNE) (ab46545), thioredoxin2 (ab26320), α-SMA (ab5694), GRP78 (ab955), XBP-1 (ab37152), TGF beta Receptor I (ab31013), TGF beta Receptor II (ab61213), and acetyl-lysine (ab80178). Antibodies against the following proteins were purchased from Cell Signaling Technology: phosphorylated (P-)Smad3 (8769), total (T-)Smad3 (9513s), and GAPDH (2118). The antibodies against P-PERK (sc-32577) and T-PERK (sc13073) were purchased from Santa Cruz Biotechnology. The antibody against ATF6 (15794-1-AP) was purchased from Proteintech Group. The secondary antibodies used in this study were acquired from LI-COR Biosciences (used at 1:10,000 dilution). The GT VisionTM+Detection System/Mo&Rb reagent for immunohistochemistry was obtained from Gene Technology (Shanghai, China). The Alexa Fluor 488-conjugated goat anti-rabbit secondary antibody for immunofluorescence was purchased from LI-COR Biosciences.

On day 5 after kidney transplantation, the kidney grafts were harvested, formalin fixed and paraffin embedded. Staining with hematoxylin and eosin (H&E), periodic acid-Schiff, anti-C3d (R&D Systems, AF2655, 1:50) and anti-IgG antibody (Bethyl Laboratories, A90-116B, 1:500). The sections were examined for severity of rejection by light microscopy. Features of inflammation examined included peritubular capillary inflammatory cell infiltration, tubular injury as measured by the presence of acute epithelial damage as well as C3d and IgG deposition were measured according to the semiquantitative Banff scoring criteria: 0, absent; 1, mild; 2, moderate; and 3, prominent. Each stain was evaluated on four complete cross-sections. For immunofluorescence analyses, 6-µm tissue sections were cut from samples embedded in O.C.T. (SkuraFinetek). After drying, sections were fixed in 100% acetone for 10 min at 4°C, and then blocked with 10% BSA at room temperature for 30 min, following by staining primary antibodies at 4°C overnight. Alexa Fluor-conjugated secondary antibodies (Life Technologies) were used to detect specific murine antigens. The antibodies were as follows: mouse monoclonal antibody against mouse CD68 (abcam, ab955, 1:200), rat monoclonal antibody against mouse MHC class II (abcam, ab25333, 1:150), rabbit polyclonal antibody against mouse CD206 (abcam, ab64693, 1:100).