After exposure to T cell-inducible cell death assay immobilized in a collagen-coated plate, floating cells (including T cells) were removed by washing with PBS. The cells were then stained for 30 min at room temperature in the dark with TO-PRO3 (1 μM, ThermoFisher Scientific) and Hoechst33342 (5 μg/ml, Molecular Probes). After washing, photomicrographs of dead cells were acquired under a Keyence Fluorescence Microscope (BZ-9000) using CFI Plan Apo ×40 0.95/0.14 mm objective lenses (Nikon). The observed signals were processed using the BZ-II image analysis application (Keyence).
Bz 2 image analysis application
Manufactured by Keyence
The BZ-II image analysis application is a software tool designed for quantitative image analysis. It provides users with the ability to capture, process, and analyze digital images. The core function of the BZ-II is to enable image-based measurements and data extraction, supporting a range of scientific and industrial applications.
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Lab products found in correlation
Hoechst 33342, by Thermo Fisher Scientific (1 mentions)
Prolong gold antifade reagent with dapi, by Thermo Fisher Scientific (1 mentions)
Lsm 710 confocal laser scanning microscope, by Zeiss (1 mentions)
Prolong gold antifade mountant, by Thermo Fisher Scientific (1 mentions)
Bz 9000 microscope, by Keyence (1 mentions)
5 protocols using bz 2 image analysis application
1
Quantifying Cell Death via Fluorescence Microscopy
2
Histological Analysis of Intestinal Radiation Damage
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At 0, 6, 72 and 120 h after radiation or poly I:C treatment, jejunal tissues were excised from small intestine 4 cm below the gastroduodenal junction, flushed with PBS, fixed in 10% formalin overnight and embedded in paraffin. Five-micrometre sections were stained with hematoxylin and eosin (H&E) and observed under a light microscope using BZ-II Image Analysis Application (BZ-9000, Keyence). Crypt survival was evaluated at 3 days after irradiation by a modification of microcolony assay17 (link). Briefly, the numbers of regenerating crypts in jejunal sections were counted based on histological appearance. Regenerating crypts were defined as those containing ≥10 adjacent chromophilic non-Paneth cells and at least one Paneth cell. Tibias were collected at 0 and 5 days after irradiation, fixed in 70% ethanol for 3 days, embedded in methyl methacrylate resin and stained with H&E.
Cell death in the small intestine was evaluated by TUNEL-staining. Paraffin sections of small intestine were treated with reagents supplied in the in situ Cell Death Detection Kit, Fluorescein (Roche Diagnostics). Samples were mounted in ProLong Gold Antifade Reagent with DAPI (Invitrogen) and observed under a fluorescence microscope using BZ-II Image Analysis Application. At least 50 crypts were counted to calculate the numbers of TUNEL-positive cells per crypt.
Cell death in the small intestine was evaluated by TUNEL-staining. Paraffin sections of small intestine were treated with reagents supplied in the in situ Cell Death Detection Kit, Fluorescein (Roche Diagnostics). Samples were mounted in ProLong Gold Antifade Reagent with DAPI (Invitrogen) and observed under a fluorescence microscope using BZ-II Image Analysis Application. At least 50 crypts were counted to calculate the numbers of TUNEL-positive cells per crypt.
3
Immunohistochemical Analysis of Adipose Tissue
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Epididymal/inguinal fat tissues were isolated from isoflurane anesthetized mice, fixed in 10% neutral-buffered formalin, and embedded in paraffin. Then, 3-µm-thick sections were cut, deparaffinized, and stained using rabbit anti-uncoupling protein-1 (UCP1) antibody (1/200 dilution; catalog No. ab10983, RRID: AB_2241462, Abcam), mouse anti-CD11c antibody (1/100; catalog No. 60258-1-Ig, RRID: AB_2881379, Proteintech), or goat anti-CD206 antibody (1/100; catalog No. sc-34577, RRID: 2144904, Santa Cruz) as primary antibodies, and Alexa Fluor 594-labeled donkey anti-rabbit immunoglobulin G (IgG) (1/200; catalog No. A21207, RRID: AB_141637, Invitrogen), Alexa Fluor 594-labeled donkey anti-mouse IgG (1/200; catalog No. A21203, RRID: AB_141633, Invitrogen), or Alexa Fluor 488-labeled donkey anti-goat IgG (1/200; catalog No. A11055, RRID: AB_2534102, Invitrogen) as secondary antibodies. The section slides were examined using an LSM710 confocal laser-scanning microscope (Zeiss). To evaluate adipocyte sizes, formalin-fixed adipose tissue sections were stained with hematoxylin/eosin, and the long axis of 300 independent mature adipocytes (per mouse) was measured by manual tracing using a BZ-II image analysis application (Keyence). Liver sections were examined by hematoxylin/eosin (HE) staining.
4
TUNEL Staining of Apoptotic Cells in Skin
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Formalin-fixed, paraffin-embedded skin sections were used for TUNEL staining. After deparaffinization, sections were treated for 1 h at room temperature with Proteinase K (10 μg/ml in 10 mM Tris-HCl, pH 7.4). The TUNEL reaction was performed using the In Situ Cell Death Detection Kit, Fluorescein (Roche). For preservation, labeled sections were mounted in ProLong Gold Antifade Mountant (ThermoFisher Scientific) containing DAPI. Fluorescence images of apoptotic cells were acquired under a Keyence Fluorescence Microscope BZ-9000 using CFI Plan Apo ×40 0.95/0.14 mm objective lenses (Nikon). The observed signals were processed using the BZ-II image analysis application (Keyence).
5
Lung Function Evaluation and Histology
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