Immunohistochemical analyses were performed on specimens from xenograft tumors. Formalin-fixed paraffin-embedded tissue sections (4μm thickness) were deparaffinized and rehydrated in graded ethanol solutions (100%, 95%, 80%, 70%). For IHC staining, after antigen repair, the tissue slides were incubated with anti-Ki-67 (1:200, #9027S, Cell signaling, USA) overnight at 4°C, then washed with PBS and incubated with a horseradish peroxidase-labelled secondary antibody for 30 min and stained with diaminobenzidine (DAB) for 5 min. For H&E staining, tissue sections were stained with hematoxylin for 3 min and eosin solution for 30 s. For the TdT-mediated dUTP nick-end labelling (TUNEL) assay, we used a TUNEL in situ apoptosis detection kit (Roche, USA) according to the manufacturer’s instructions. The stained images were observed using a fluorescence microscope at a 200-fold magnification.
Tunel in situ apoptosis detection kit
Manufactured by Roche
Sourced in United States, Switzerland
The TUNEL in situ apoptosis detection kit is a laboratory tool used to identify and quantify apoptotic cells in tissue samples. It utilizes the terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) method to detect DNA fragmentation, a hallmark of apoptosis. The kit provides reagents and materials necessary to perform this analysis.
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4 protocols using tunel in situ apoptosis detection kit
1
Immunohistochemical Analysis of Xenograft Tumors
2
In Vivo Apoptosis Detection Using TUNEL Assay
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Apoptosis in vivo was detected using the terminal deoxytransferase-mediated dUTP nick end labeling (TUNEL) in situ apoptosis detection kit (Roche) according to the manufacturer’s instructions. After deparaffinization and dehydration and inactivation of peroxidase activity, 20 paraffin sections of each tumor specimen were incubated with 2 μg/mL proteinase K at 37 °C for 15 min. Then, the sections were treated with terminal deoxynucleotidyl transferase (TdT) and biotinylated dUTP, and observed under microscopy. Controls were treated in the same manner except that TdT was replaced with dH2O.
3
TUNEL Assay for Retinal Apoptosis
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Apoptosis was detected using the terminal deoxytransferase-mediated dUTP nick end-labeling (TUNEL) in situ apoptosis detection kit (Roche Diagnostics, Mannheim, Germany), according to the manufacturer’s instructions. Briefly, paraffin cross-sections were deparaffinized and rehydrated. The tissue samples were incubated for 6 min at room temperature with proteinase K working solution. After PBS washing, tissues were treated with TUNEL reaction mixture and incubated for 60 min at 37°C in a humidified atmosphere. Slides were then rinsed three times in PBS at room temperature for 5 min and counterstained with DAPI. Only TUNEL-positive cells in retinal ganglion cell layer were counted. For each time point, eight eyes were used for statistical analysis. The number of TUNEL positive cells per eye was calculated from the average number of four central retinal sections.
4
Dextran Sulfate Sodium-Induced Colitis Model
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The chemicals utilized in this study were as follows: dextran sulfate sodium (DSS; MP Biomedicals, LLC, Solon, OH, USA; lot number: BJ14745, molecular weight: 5000); berberine hydrochloride (BBR; Shanghai Yuanye Bio-technology Co., Ltd., Shanghai, China; lot number: Y18D8C50814, molecular weight: 371.81); glucose-regulated protein 78 (GRP78), caspase-12, and caspase-3 primary antibodies (Abcam, Cambridge, UK; lot numbers: GR309483-1, ab62484, and ab13847, respectively); anti-rabbit and anti-mouse secondary antibody (Beijing ZSGB-BIO, Beijing, China; lot numbers: K167722B and K175212C, respectively); proteinase K (Beijing Tiangen, Beijing, China; lot number: M2011); DAB chromogenic reagent kit (Beijing ZSGB-BIO, lot number: K167722A); TUNEL in situ apoptosis detection kit (Roche, Basel, Switzerland; lot number: 11906800); high-purity total RNA rapid extraction kit (Shanghai Generay, Shanghai, China; lot number: 1703G01); HiScript-II Q RT SuperMix for qPCR reverse transcription kit (Nanjing Vazyme, Nanjing, China; lot number: 7E092G6); and ChamQ SYBR Color qPCR Master Mix (Nanjing Vazyme Co.; lot number: 7E092H6).
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