Biozero microscope

Paraffin-embedded sections of female alates were used to observe the frontal glands, normally restricted to the heads^{40 (link),41 (link)}. Those of heads of induced presoldiers and natural soldiers were used to observe the frontal glands. Samples preserved in 70% ethanol were dehydrated in increasing concentrations of ethanol, transferred into xylene, and embedded in paraffin. Serial parasagittal sections (6 μm thick) were processed using an MRS80-074 microtome (Ikemoto, Tokyo, Japan) and stained with hematoxylin and eosin. Tissues on the slides were observed under a Biozero microscope (Keyence).

One milliliter of 0.5% agarose (Lonza Japan) in RD medium was solidified in each well of a 6-well plate (Corning, NY, USA) as the bottom agarose layer and incubated for 20 min at 24°C. The 0.3% top agarose containing cells was poured over the 0.5% agarose gel (2,500 cells/well) and set at 10 min at 4°C. The plate was cultured in a humidified incubator at 37°C with 5% CO₂ for 14 days, rinsed twice with PBS, and fixed with methanol for 15 min. Colonies were stained for 20 min with Giemsa’s solution (Merck, Darmstadt, Germany) diluted 1: 20 in phosphate buffer (4.7 mM KH₂PO₄, 2 mM Na₂HPO₄). Digital images of the colonies were taken using a Biozero microscope (Keyence). Colonies were counted using Image J software (NIH, USA).

Deparaffinised sections were digested with proteinase (Dako Denmark A/S, Glostrup, Denmark) for 10 min and treated with 3% hydrogen peroxide (Wako Pure Chemical Industries Ltd., Osaka, Japan) to block endogenous peroxidase activity. After antigen retrieval, the sections were incubated overnight at 4 °C with primary antibodies against the following mouse proteins: matrix metalloproteinase-13 (MMP-13, 1:100, Abcam, Cambridge, UK), A Disintegrin And Metalloproteinase with Thrombospondin motifs-5 (ADAMTS-5, 1:100, Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA), IL-6 (1:100, Abcam), type II collagen (1:100, Abcam), IL-1β (1:100, Abcam), inducible nitric oxide synthase (iNOS, 1:100, Abcam), Cluster of Differentiation 206 (CD206, 1:100, Abcam), and human nuclear antigen (1:100, Abcam). Sections were then incubated with peroxidase-labelled anti-rabbit or mouse immunoglobulin (Histofine Simple Stain MAX Po; Nichirei Bioscience, Tokyo, Japan) at room temperature for 30 min. Staining was visualised using the peroxidase substrate, 3,3′-diaminobenzidine, which resulted in the development of a brown-coloured signal, with methyl green used for counterstaining. As negative controls, a non-immune mouse or rabbit immunoglobulin (1:50 dilution) was used instead of the primary antibodies. Images were acquired using a Biozero microscope (Keyence Corp., Itasca, OH, USA).

Cryosections of 3.7% neutral-buffered formalin-fixed tumors were stained with the following antibodies: CD45 rat anti mouse 1:50 (BD Pharmingen; clone: 30F11); CD11b rat anti mouse 1:50 (BD Pharmingen; clone: M1/70); and F4/80 rat anti mouse 1:50 (eBioscience; clone: BM8). The secondary antibody used was goat anti rat Alexa 594 1:500 (Dianova #112-585-062). Active caspase 3 rabbit anti mouse 1:100 (Abcam, #ab2303) combined with goat anti rabbit Cy2 1:500 (Dianova, #111-227-003) was used to determine apoptosis in tumors.
To determine the degree of liver fibrosis, 5 μm frozen sections were stained. Sections were fixed in 4% paraformaldehyde. Extracellular matrix was stained using picrosirius red. Collagen type I was stained using rabbit anti collagen-type-I (Millipore, #AB765P); Cre-recombinase was stained using rabbit anti cre (Novagen, #69050–3) and goat anti-rabbit conjugated with Cy2 (Dianova, #111-227-003).
Sections were photographed using a Keyence Biozero microscope (Keyence, Germany) and processed using ImageJ. Quantification was performed in at least three sections per mouse in at least three mice per group or more, as noted in the figure legends. At least 0.9 mm² was examined per section.

Microscopy study of direct internalization: HEK293 and RH7777 cells overexpressing human ASCT2‐GFP were used. Cells were incubated with Ab3‐8 (10 μg/mL) for 1 hour at 37°C. Fluorescence images were obtained using a BioZero microscope (Keyence) with a 20 × Plan Fluor objective lens. We observed at least 20 cells in each cell line. Quantification analysis using FCM: Cells (2 × 10⁶) were reacted with Ab3‐8 (10 μg/mL) at 4°C for 30 minutes. After that, the cells were divided into two groups; one was incubated at 4°C, and the other did at 37°C for 1 hour. Cells were reacted with PE‐labeled anti‐rat IgG at 4°C for 1 hour, followed by FCM analysis.

The tissue array was purchased from BioChain, Newark, CA. Acetone-fixed sections were blocked in 10% normal goat serum and then incubated with mouse anti-CD239 (87207) and rabbit anti-HER2 (29D8) mAbs. Both antibodies were diluted with Ca²⁺- and Mg²⁺-free phosphate-buffered saline (PBS(−)) containing 1% bovine serum albumin (BSA); all washes were performed in PBS(−). Secondary antibodies conjugated to Alexa Fluor 488 or 594 were used. After several washes, sections were mounted in 90% glycerol containing 0.1× PBS and 1 mg/mL p-phenylenediamine. Images were captured using a Biozero microscope (Keyence, Osaka, Japan). The scaling of HER2 expression was performed according to the guidelines^{28 (link)}. The expression of CD239 was defined similarly. Strong (+++) is based on circumferential membrane staining that is complete and intense. Moderate (++) is defined by circumferential membrane staining that is incomplete and/or weak/moderate. Weak (+) is based on incomplete membrane staining that is faint. N.D. (−) is negative staining.