Immunohistochemical staining (IHC) on tumor sections was performed as described previously (32 (link)–34 (link)), using antibodies against EPHA2 (Life Technologies; #347400), EGFRL858R (Cell Signaling Technology, #3197S), PCNA (BD Pharmingen, #555567), and vWF (DakoCytomation, #A0082). PCNA+ staining was quantified as the average percentage of PCNA+ nuclei relative to total nuclei (proliferation index). Apoptosis assays were performed using the Apoptag Red In Situ Apoptosis Detection Kit per the manufacturer’s protocol (Millipore). TUNEL+ staining was quantified as the percentage of TUNEL+ nuclei relative to total nuclei (apoptotic index). Tumor vessels were quantified by assessing the vWF+ vessels (pixels). Four fields of at least 5 independent tumors per genotype or treatment condition were assessed for all staining quantification. Biotin goat anti-rabbit (BD Pharmingen), anti-rabbit Cy3 (Jackson ImmunoResearch), retrievagen A (pH 6.0) (BD Pharmingen, #550524), streptavidin peroxidase reagents (BD Pharmingen, #51-75477E), and the liquid 3,3′-diaminobenzidine tetrahydrochloride substrate kit (Zymed Laboratories) were used for IHC. Cytoseal XYL (Richard Allan Scientific) or ProLong Gold antifade reagent with DAPI (Life Technologies) were used to mount slides.
Apoptag red in situ apoptosis detection kit
Manufactured by Merck Group
Sourced in United States, Germany, France
The ApopTag Red In Situ Apoptosis Detection Kit is a laboratory equipment product used to detect and visualize apoptosis, a form of programmed cell death, in tissue samples. The kit utilizes terminal deoxynucleotidyl transferase (TdT) to label DNA strand breaks, which are characteristic of apoptotic cells. The labeled DNA is then detected using a red fluorescent dye, allowing for the identification and quantification of apoptotic cells within the sample.
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Lab products found in correlation
Lsm 710 confocal microscope, by Zeiss (12 mentions)
Vectashield, by Vector Laboratories (6 mentions)
Hoechst 33342, by Thermo Fisher Scientific (6 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (5 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (5 mentions)
Apoptag kit, by Merck Group (4 mentions)
Lsm 710, by Zeiss (4 mentions)
Lsm 5 pascal, by Zeiss (3 mentions)
Tcs sp5 confocal microscope, by Leica (2 mentions)
Anti chicken af488, by Thermo Fisher Scientific (2 mentions)
Anti gfp, by Abcam (2 mentions)
Axio imager m2, by Zeiss (2 mentions)
Dmi8 automated microscope, by Leica (2 mentions)
Fluoromount dapi, by Thermo Fisher Scientific (2 mentions)
Proteinase k, by Roche (2 mentions)
Triton x 100, by Merck Group (2 mentions)
Apoptag peroxidase in situ apoptosis detection kit, by Merck Group (2 mentions)
Lsm 780, by Zeiss (2 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (2 mentions)
Axiovert 200, by Zeiss (2 mentions)
172 protocols using apoptag red in situ apoptosis detection kit
1
Quantitative Analysis of Tumor Biomarkers
2
TUNEL Assay for Apoptosis in Zebrafish Retina
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Terminal deoxynucleotide transferase (TdT)-mediated dUTP nick labeling (TUNEL) was performed on 3 dpf retinal cryosections using the ApopTag Red In Situ Apoptosis Detection Kit (Millipore, Temecula, CA) according the manufacturer’s instructions and co-labeled for rods (4C12) to identify mutants. TUNEL positive cells were counted in the ONL, INL and GCL from: WT (n = 7); ljr (n = 9); control (n = 7) and six7-MO1 (n = 6), one section for individual embryo. TUNEL positive cell counts were transformed (log Y+1) before student t test was conducted. TUNEL assay was performed in six7fl4 mutants and WT embryos at 56 hpf and 4 dpf. Tail-clip genotyping was used to identify mutants at 56 hpf. The following strains and number (n) of 56 hpf were analyzed: WT (n = 5), six7fl4 (n = 7) and at 4 dpf: WT (n = 3), six7fl4 (n = 6).
3
TUNEL Assay for Apoptosis Detection
4
Detecting Apoptosis in Transgenic Zebrafish
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Transgenic Tg(-8.0cldnb:lynEGFP) embryos developed to 30–32 hpf were fixed in 4% PFA at 4°C overnight. TUNEL assay was performed using a kit as previously described (ApopTag Red in situ Apoptosis Detection kit, Millipore; Xu et al., 2014 (link)). The presence of TUNEL+ cells (red fluorescence in the pLLP) was analyzed under a fluorescence microscope.
5
Liver Regeneration in Zebrafish Larvae
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Zebrafish larvae were incubated in 10 mM BrdU (Sigma B5002) for 12 h before fixing. After the incubation, the larvae were immediately washed six times in E3 medium for 1 h. The samples were then fixed in 4% PFA for 4 h at room temperature. After 2 h, we dissected the liver from the whole larvae and immunostaining was performed as described previously (Wang et al., 2012 (link)) using the following antibodies: BrdU mouse antibody Alexa Fluor 647 (Thermo Fisher, B35133, 1:2000) and Hoechst 33342 (Thermo Fisher, H3570, 1:5000). TUNEL assays were carried out using the ApopTag Red in situ Apoptosis Detection Kit (Millipore, S7165).
6
Quantifying Apoptosis in Wing Discs
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Third instar larvae were dissected in PBS pH 7.6, fixed in PBS/formaldehyde 3.7%, washed three times for 10 min in PBT (1 × PBS, 0.5% Triton). Discs were then dissected and TUNEL staining was performed according to manufacturer's instructions (ApopTag Red in situ apoptosis detection kit, Millipore, Temecula, CA, USA). Discs were mounted in CitifluorTM (Biovalley, Marne-La-Vallée, France) and observed with a Leica SPE upright confocal microscope (Leica, Wetzlar, Germany). White patches in the wing pouch were counted for at least 30 wing imaginal discs per genotype. Student's t-tests were performed and results were considered to be significant when α< 5%.
7
Visualizing and Quantifying Apoptosis in Zebrafish
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Apoptosis were detected with TUNEL assay. Embryos were harvested at the indicated stages, fixed in 4% PFA, incubated in 1μg/ml Proteinase K solution to solubilize tissues and rinsed twice with PBS-Tween. Apoptotic cells were labeled using ApopTag® Red In Situ Apoptosis Detection Kit (Millipore, Billerica, MA, USA) following the manufacture’s instruction. Fluorescent images were taken with a LSM 710 confocal microscope from Carl Zeiss. To detect the activity of caspase 3 in zebrafish, embryos were collected at 60 hpf and homogenated in ice-cold RIPA buffer. Then, the homogenate was mixed with an equal volume of 0.39 mM of Acetyl-Asp-Glu-Val-Asp-7-amido-4-methylcoumarin (Sigma-Aldrich, Saint Louis, MO, USA), a fluorogenic substrate for caspase 3. Quantification of caspase 3 activity was performed by fluorescent detection of free AMC (also known as 7-amino-4-methylcoumarin) as described previously [43 (link)].
8
TUNEL Assay for Apoptosis Detection
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ApopTag Red In Situ Apoptosis Detection Kit (Millipore) was used for the TUNEL assay. Animals were fixed as for in situ hybridization, incubated overnight at 37°C in terminal deoxynucleotidyl transferase diluted in reaction buffer, washed in stop/wash buffer, rinsed in PBST (0.3% TritonX), and blocked for 30 min in PBSTx containing 5% heat-inactivated horse serum and 5% Western Block Reagent (Roche). Animals were then developed in anti-digoxigenin rhodamine conjugate diluted in block solution overnight at 4°C in the dark.
9
Detecting Neuronal Cell Death via TUNEL
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Neuronal cell death was detected by terminal deoxyuridine triphosphate nick end labeling (TUNEL) (ApopTag Red In Situ Apoptosis Detection Kit, Millipore), according to the manufacturer’s protocol, followed by labeling with NeuN and DAPI. Cells triple-positive for TUNEL, NeuN, and DAPI were considered as neurons undergoing cell death.
10
Immunohistochemical Analysis of Tumour Tissues
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The tumour tissues dissected from tumour-bearing mice were subjected to immunohistochemistry analysis using methods described in our previous publication 80 (link). The antibodies used include mouse anti-human Ki-67 antibody (Abcam, Cat No: ab15580, 1:100 dilution) and goat anti-mouse HRP-labelled secondary antibody (Thermo Fisher, Cat No: 31430) as well as DAB peroxidase substrate solution (Vector Laboratories, Burlingame, CA). For performing the TUNEL assay, the indicated slides were performed using the ApopTag Red In Situ Apoptosis Detection Kit (Millipore) according to the manufactural instruction.
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