Anti cd3

Formalin-fixed and paraffin-embedded tissues sectioned to 4 μm were used for histological evaluation of liver tumors in a mouse model. Hematoxylin and eosin (H&E) staining was performed for each sample. For IHC, tissue slides were deparaffinized with xylene and rehydrated through a graded series of ethanol solutions (100%, 95%, and 70%). Subsequently, the slides were subjected to antigen retrieval by microwaving in a citric acid solution for 15 min. The primary antibodies anti-hepatocyte (Abcam, Cat#ab75677), anti-Epcam (Abcam, Cat#ab213500), anti-CD3 (Abcam, Cat#ab16669), anti-Ly6G (Servicebio, Cat#GB11229), anti-CD8 (Abcam, Cat#ab217344), anti-F4/80 (CST, Cat#70076), anti-Asma (Servicebio, Cat#BM0002), and anti-CD31 (Servicebio, Cat#GB113151) were used. Subsequently, the slides were incubated with secondary antibodies (1:1, 100 μL for each slide; HRP-anti-rabbit IgG, ZSGB, Cat#PV-6001, or HRP-anti-mouse IgG, ZSGB, Cat#PV-6002) for 10 min at room temperature. Multispectral images were scanned with a ZEISS AXIOSCAN 7 instrument. Cells of interest were quantified in Halo v3.4 (Indica Labs) or QuPath v0.2.0. Each section was evaluated by 2 or 3 experienced pathologists.

Mouse femoral bones were fixed in 4% PFA for 2 h at 4 °C, decalcified with 0.5 M EDTA (Sigma-Aldrich) for 4 weeks, and decalcified bones were immersed in a 20% sucrose and 2% polyvinylpyrrolidone (Sigma-Aldrich) solution for 1 week.
For immunostaining, the bones were embedded and frozen in OCT, and 6-μm-thick sections were cut along the longitudinal axis of the femur. Bone sections were blocked in 5% bovine serum albumin and 0.3% Triton X-100 for 1 h, and incubated with antibodies (1:300 for primary antibodies, 1:1000 for secondary antibodies). Nuclei were counterstained with DAPI. The antibodies were as follows: anti-CD3 (abcam, Cambridge, MA), anti-RANKL (abcam), Alexa Fluor 555 Donkey Anti-Rabbit IgG H&L, and Alexa Fluor 488 Goat Anti-Rat IgG H&L (abcam).
To detect osteoblasts and osteoclasts, the tissues were dehydrated in 75% ethanol overnight, then sequentially in 85% ethanol, 95% ethanol, and 100% ethanol for 1 h each. The femoral bones were treated with xylene twice for 15 min, then embedded in paraffin, and 5-μm-thick sections were taken. Morphological analysis of osteoblasts was performed by standard hematoxylin and eosin (H&E) staining. TRAP was stained using an acid phosphatase assay kit (Sigma-Aldrich), and TRAP-positive osteoclasts were examined at 20× magnification.

FFPE tissue sections were deparaffinized by incubation in a 60 °C oven for 30 min and in Xylene for 10 min. Hydration was achieved by sequentially immersing slides for 10 min each, in staining dishes containing 100%, 95%, 80% ethanol, and ddH2O. Antigens were unmasked by boiling slides in 1× Sodium citrate solution (10 mM Sodium citrate, pH 6.0) in a pressure cooker for approximately 20 min (first 10 min without weight at 350 °C and next 10 min with weight at 240 °C). After antigen retrieval, tissues were blocked in 3% H2O2 in phosphate buffered saline (PBS) for one hour at room temperature to quench endogenous peroxidase. To quench non-specific antibody binding, the tissue sections were blocked in horse serum for an hour. Tissues were then stained with primary antibodies overnight in a humidity chamber at 4 °C. Following overnight incubation, the tissues were washed in TBST three times 30 min each before an hour of incubation with secondary antibodies. The Vectastain Elite ABC-HRP Kit RTU (Vector Laboratories, PK-7200) was used to develop the peroxidase before incubating with the ImmPACT-DAB chromogen. The primary antibodies used were Ki-67 (dilution 1:800, clone D3B5, Cell Signaling Technology, CST 12202S) and anti-CD3 (dilution 1:500, Abcam, ab 16669, Rabbit mAb, SP7).

Lung sections of Sham, CIA, ODE, and CIA+ODE treatment groups previously obtained [11 (link)] were stained with H&E or with anti-CD3 (1:100, Cat#ab5690, Lot#GR3356033-2), anti-CD68 (1:50, Cat#ab31630, Lot#GR3305929-3), and anti-MPO (1:25, Cat#ab9535, Lot#GR331736-4) from Abcam (Cambridge, MA), anti-CD45R (1:40, Cat#14-0452-82, Lot#2178350) from Invitrogen (Grand Island, NY), and anti-CCR2 (1:100, Cat# NBP267700, Lot# HMO537) from Novus Biologicals (Centennial, CO). Cross absorbed (H+L) goat anti-rabbit (Cat#A32731, Lot#UK290266), goat anti-mouse (Cat#A32727, Lot#UL287768) and goat-anti rat (Cat#A21434, Lot#2184321) from Thermo Fisher, Grand Island, NY) were used at 1:100 dilution as secondary antibodies. Slides were mounted with VECTASHIELD® Antifade Mounting Medium with DAPI (Cat#H-1200, Lot#ZG1014, Burlingame, CA) and visualized under Zeiss fluorescent microscope.

3 μm formalin-fixed, paraffin-embedded sections were prepared and stained, as previously described (28 (link)). Cell nuclei were stained with DAPI (Hoechst 33342, Molecular Probes H-1399, Netherlands). Primary antibodies used were anti-rat IgG-Alexa Fluor 647 (ThermoFisher, A21472) for plasma cells, anti-IL-21 (Bioss bs-2621R-a350, Boston, USA), anti-CD20 (Santacruz sc-393894, Dallas, USA) for B cells, anti-Ki67-Fitc (ThermoFisher 11-5698) for proliferating GC cells, and anti-CD3 (Abcam 5690, Cambridge, UK) for Tfh, which were counted within Ki67⁺ GC (activated Tfh) and the MZ (resting Tfh) regions as demonstrated in Figure 4A. Secondary antibodies used were donkey anti-rabbit-biotin (Dianova, 711-065-152, Hamburg, Germany) and AlexaFluor594 Tyramide SuperBoost Kit, strepavidin (ThermoFisher B40935) for anti-IL-21, goat anti-mouse-IgM-Cy3 (Dianova 115-166-075) for anti-CD20 and donkey anti-rabbit-Cy5 (Dianova 711-175-152) for anti-CD3. Digital pictures were assessed using Histoquest^® software.