Normal goat placenta cotyledons (45-60 days of pregnancy) were isolated sterile tactically according our previous study 23 (link), then the cotyledons were minced into about 5 mm3 pieces and incubated in completed DMEM/F12 culture medium supplemented with 2.4 μg/mL of SW for 48 hr, followed by 4% paraformaldehyde fixation and paraffin-embedding. Cotyledons pieces cultured in completed DMEM/F12 culture medium served as negative control. To assess apoptosis in SW treated goat placenta cotyledons, a TUNEL assay was performed according to the manufacturer's instructions (Vazyme, Piscataway, NJ, USA). In brief, paraffin embedded sections were pretreated with 20 μg/mL proteinase K, underwent antigen retrieval, blocked with 10% normal goat serum, and then stained for terminal deoxynucleotidyl transferased UTP nick end labeling using a reaction mixture containing tetramethyl rhodamine-dUTP (TMR red-dUTP). GTCs were labeled by immunofluorescence using cytokeratin 7 (CK-7) antibodies according to our previous study 23 (link). Finally, nuclei were counterstained with DAPI and observed with laser confocal scanning microscopy (A1R/A1, Nikon, Japan).
Tunel assay
Manufactured by Vazyme
Sourced in China, United States
The TUNEL assay is a method used to detect and quantify apoptosis, a programmed cell death process. The assay works by labeling the fragmented DNA that is characteristic of apoptotic cells, allowing for the identification and analysis of these cells.
Automatically generated - may contain errors
Lab products found in correlation
Lsm800 confocal microscope, by Zeiss (4 mentions)
Hoechst 33342, by Thermo Fisher Scientific (2 mentions)
Lsm 700 confocal microscope, by Zeiss (2 mentions)
A1r a1, by Nikon (1 mentions)
Cell counting kit 8 assay, by Solarbio (1 mentions)
Von kossa staining, by Solarbio (1 mentions)
Bx53 fluorescence microscope, by Olympus (1 mentions)
Hoechst 33258, by Thermo Fisher Scientific (1 mentions)
12 protocols using tunel assay
1
Apoptosis Assessment in Goat Placenta Cotyledons
2
Tissue Apoptosis Quantification by TUNEL
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Tissue fraction was determined by TUNEL assay. The TUNEL assay was performed according to the instruction manual (Vazyme Biotech, China). Images were photographed by fluorescent microscopy at 400× magnification.
3
TUNEL Assay on Mouse Testis Slices
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Mouse testicle slices were used for the TUNEL assay, which was conducted according to the manufacturer’s instructions (Vazyme Biotech, China).
4
Apoptosis Quantification via TUNEL Assay
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The TUNEL assay in cells and tissues was performed according to the manufacturer’s instructions (Vazyme Biotech Co., Ltd, China). For cells, A2780 and ID-8 cells were treated with Tan-I (4.8 µg/mL), Paclitaxel (0.1 µg/mL), or Tan-I combined with Paclitaxel for 24 hours. The cells were first incubated in a bright red labeling mix for 45 minutes at 37 °C, followed by treatment with Click-iT reaction cocktail. The nucleus was stained with DAPI. For tissues, the sections of tumor tissue were deparaffinized in xylene and rehydrated in PBS buffer. The sections were incubated with Biotin-dUTP Labeling Mix for 1 hour at 37 °C in the dark and then were covered in Streptavidin-HRP for 30 minutes. The slides were visualized by the DAB substrate and looked at using a microscope (OLYMPUS, Japan). TUNEL-positive cells and the apoptotic index was calculated as a ratio of (apoptotic cell number)/(total cell number) in each field.
5
Tent5a Knockdown Effects on Myoblasts
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C2C12 cells at 70% confluence were selected and prepared into a single‐cell suspension. To detect the effect of Tent5a knockdown on myoblast viability, C2C12 cells were inoculated in 96‐well plates, and 2000 cells in 100 µl of growth medium were seeded in each well. After incubation for 24 h, the Cell Counting Kit‐8 assay (YZ‐CK04, Solarbio) was performed according to the manufacturer's protocol. The wound‐healing assay was used to quantify cell migration. Cells were inoculated with a 6‐well plate and scratched when the cell density increased close to 100%. The observation time points of the wound‐healing assay included 0, 24, and 48 h. To detect the effect of Tent5a knockdown on myoblast apoptosis, C2C12 cell apoptosis was induced by H2O2, and a TUNEL assay (A113, Vazyme) was conducted according to the manufacturer's protocol. After culturing in differentiation medium for 5 days, C2C12 cells were fused and formed mature myotubes. Immunofluorescence staining and qPCR were used to detect the effect of Tent5a knockdown on myoblast differentiation.
6
Histological Analysis of Mouse Testes
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Mouse testes or epididymis were collected from at least three mice for each genotype. The tissues were fixed in modified Davidson’s fluid for up to 24 h and stored in 70% ethanol. The samples were then dehydrated through a graded ethanol series and embedded in paraffin. Tissue sections (thickness five mm) were prepared and mounted on glass slides. After deparaffinization, slides were stained with periodic acid Schiff for histological analysis. Apoptotic cells in testis were detected using the terminal deoxynucleotidyl transferase dUTP nick end-labeling (TUNEL) assay (Vazyme, Nanjing, China) according to the manufacturer’s instructions.
7
TUNEL Assay for Apoptosis in Mouse Testis
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DNA fragmentation was determined as an index of apoptosis in paraffin-embedded mouse testis sections from three Tulp2–/– mice and three WT mice via the terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay (Vazyme, A113-01) according to the manufacturer’s specifications. Briefly, the testis sections were washed in PBS and equilibrated with TdT buffer for 20 min at room temperature. TdT buffer was removed and terminal transferase reaction mix was added. The reaction was performed for 1 h at 37°C. Sections were washed with PBS and counterstained with Hoechst. All spermatogenesis tubules (approximately 200 tubules) from each section from each mouse were analyzed using a LSM700 confocal microscope (Zeiss, Germany). The nuclei of TUNEL-positive cells show red fluorescence. According to the stage of spermatogenesis and the nuclear morphology of spermatogenic cells, apoptotic cells were classified and counted by two researchers using double-blind.
8
Histological Analysis of NAFLD Progression
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Fresh liver sections were fixed in 10% paraformaldehyde for 24 h, embedded in paraffin and cut into 2 μm-thickness slices. Slices were then stained with hematoxylin and eosin (H&E) and Sirius Red. We applied a NAFLD activity score (NAS) system to estimate the degree of NAFLD progression as previously described [78 (link)]. Fibrosis index was evaluated based on Sirius Red staining according to NAS.
Frozen liver sections in 4 μm-thickness were fixed in 4% neutralized formaldehyde and stained with Oil Red-O, and the area of lipid droplet was calculated to assess hepatic lipid accumulation by ImageJ.
Hepatocyte death was detected by a terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay (Vazyme, Nanjing, China) according to the protocol. The percentage of positive cells was counted using Image J.
Frozen liver sections in 4 μm-thickness were fixed in 4% neutralized formaldehyde and stained with Oil Red-O, and the area of lipid droplet was calculated to assess hepatic lipid accumulation by ImageJ.
Hepatocyte death was detected by a terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay (Vazyme, Nanjing, China) according to the protocol. The percentage of positive cells was counted using Image J.
9
Apoptosis and Crystal Deposition in Kidneys
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Apoptoses of the kidney tissues were assessed with TUNEL assay, following the manufacturer’s instructions (Vazyme). Images were captured using a BX53 fluorescence microscope (Olympus, Tokyo, Japan). The crystal depositions in the kidneys were analyzed by Von Kossa staining according to manufacturer’s instructions (Solarbio, Beijing). The stained tissues were observed by microscopy (Olympus, Japan).
10
Immunohistochemical Analysis of Apoptosis
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Tissue sections and sperm samples were deparaffinized, rehydrated, and antigen retrieval was conducted in sodium citrate buffer by boiling samples for 10 min. Samples were cooled and blocked with 5% BSA in PBS for 2 h, followed by overnight probing with primary antibodies (Table S1 ) at 4 °C. Samples were then washed thrice using PBST (0.05% Tween 20 in 1× PBS), probed with secondary antibodies (Table S2 ), and counterstained at room temperature with Hoechst 33342 (1:1,000; Invitrogen, CA, USA) for 2 h. After an additional wash, sections were mounted, and apoptotic cells were detected via the terminal deoxynucleotidyl transferase dUTP nick end-labeling (TUNEL) assay (Vazyme) based upon provided directions (22 ,23 ). An LSM800 confocal microscope (Carl Zeiss AG, Jena, Germany) was used to image and evaluate samples.
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