Coronal brain slices prepared as described above were blocked and incubated with an anti-Iba1 (1:500; Abcam, ab178846) or anti-β-amyloid (Aβ) (1:500; Abcam, ab32136) antibody overnight at 4 °C and then with Alexa Fluor 488-conjugated goat anti-rabbit IgG (1:1000, Life Technologies) for 1 h at room temperature and counterstained with 4ʹ,6-diamidino-2-phenylindole (DAPI). Fluorescence signals were visualized with a laser scanning confocal microscope (Zeiss, Oberkochen, Germany). To measure hippocampal apoptosis, brain sections were subjected to TUNEL staining (Thermo Fisher) and counterstained with DAPI following the manufacturer’s instructions.
Tunel staining
Manufactured by Thermo Fisher Scientific
Sourced in United Kingdom
TUNEL staining is a method used to detect and quantify apoptosis, or programmed cell death, in cells. It involves labeling the fragmented DNA that is characteristic of apoptosis, allowing for the visualization and analysis of apoptotic cells.
Automatically generated - may contain errors
Lab products found in correlation
Alexa fluor 488 conjugated goat anti rabbit igg, by Thermo Fisher Scientific (1 mentions)
Ab178846, by Abcam (1 mentions)
Ab32136, by Abcam (1 mentions)
Confocal laser scanning microscope, by Zeiss (1 mentions)
Ab211091, by Abcam (1 mentions)
Ab102526, by Abcam (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions)
Dm4b microscope, by Leica (1 mentions)
4 protocols using tunel staining
1
Immunofluorescence and TUNEL Staining of Coronal Brain Slices
2
Apoptosis Assessment Utilizing TUNEL, MTT, and LDH
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Apoptosis in tissue and cell was examined using TUNEL staining (Thermo Fisher Scientific, Inc.) according to the manufacturer's instruction 42 . Besides, MTT assay and LDH release assay were determined via commercial kits (No. ab211091 and ab102526) purchased from Abcam, Inc.
3
Quantifying Liver Cell Apoptosis and Immune Profiles
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Formalin-fixed paraffin-embedded (FFPE) liver sections stained with H&E were provided by the Research Histology Facility, Imperial College London. Hepatocyte apoptosis was confirmed in FFPE liver sections using TUNEL staining (Thermo Fisher Scientific, UK), according to manufacturer’s instructions and co-staining with albumin and CD8 (online supplemental table 1 ). Immunofluorescent staining of optimal cutting temperature compound (OCT)-fixed liver cryosections was performed to assess the expression of CD8, F4/80, CD11b, CCR2, granzyme B (GZMB) using fluorochrome-labeled mAbs listed in online supplemental table 1 . Slides were mounted with fluoroshield with DAPI (Sigma-Aldrich, USA). Images were captured using the Leica DM4 B microscope and the LAS X 3.3.3.16958 software (Leica Camera AG, Germany).
4
Apoptosis Assessment in Tumor Samples
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To assess the level of apoptosis in the tumors, TUNEL staining (Thermo Fisher Scientific) was performed. Briefly, tumor samples were embedded in paraffin, sliced into 5 μm sections, which were then dewaxed, rehydrated and treated with proteinase K working solution for 30 min and subsequently with treated with H2O2 for 10 min at room temperature after a rinse with PBS. After another rinse with PBS, the slices were then treated with TUNEL reaction liquid at 37°C for 1 h in a dark humidified chamber. The sections were then washed and treated with HRP-labeled streptavidin and diaminobenzidine (DAB) for color development, with a microscope (Olympus, Tokyo, Japan) for observation.
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