Mouse pancreases were mounted, sectioned and immunostained as previously described, with minor modifications (Tonne et al., 2015 (link)). Antibodies used for immunostaining are listed in Table S2 . Briefly, 7 µm pancreas sections were fixed in 4% paraformaldehyde, permeabilized in 0.5% Triton X-100 and blocked with 5% FBS in PBS. Insulin+ cells were stained by Dako guinea pig anti-insulin antibody (1:400; Agilent Technologies). BrdU+ β-cells were stained by rat anti-BrdU antibody (1:100; Abcam), visualized by fluorescent confocal microscopy and counted manually. Proliferating β-cells were counted as insulin+ cells co-localized with BrdU and 4′,6-diamidino-2-phenylindole (DAPI) staining. Insulin+ area was visualized via horseradish peroxidase (HRP) staining using a DAB Peroxidase (HRP) Substrate Kit (Vector Laboratories). HRP slides were then imaged using an Aperio ScanScope AT Turbo Scanner (Leica Biosystems). β-cell area was measured using ImageJ (ver. 1.51u) as the percentage of insulin+ area as visualized by HRP staining in the entire pancreas section. β-cell apoptosis was determined using a TMR Red In Situ Cell Death Detection Kit (Roche). Apoptotic β-cells were counted as insulin+ cells co-localized with Tdt-mediated dUTP-X nick end labeling (TUNEL) (TMR Red In Situ Cell Death Detection Kit, Roche) and DAPI staining.
Tmr red in situ cell death detection kit
Manufactured by Roche
Sourced in Switzerland, United States, Germany
The TMR red in situ cell death detection kit is a laboratory product designed to detect and analyze cell death in various biological samples. The core function of this kit is to provide a fluorescent-based method for the identification and quantification of cells undergoing apoptosis or other forms of programmed cell death.
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Lab products found in correlation
Celltitor glo assay, by Promega (5 mentions)
Dnase 1, by Roche (2 mentions)
Vectashield, by Vector Laboratories (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Vector Laboratories (2 mentions)
Polysine slide, by Thermo Fisher Scientific (1 mentions)
Fluorescein isothiocyanate (fitc), by Jackson ImmunoResearch (1 mentions)
Hydromount, by National Diagnostics (1 mentions)
Alexa fluor 488, by Jackson ImmunoResearch (1 mentions)
Cyanine3 (cy3), by Jackson ImmunoResearch (1 mentions)
Alexa fluor 647, by Jackson ImmunoResearch (1 mentions)
Nonyl acridine orange, by Thermo Fisher Scientific (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by AnaSpec (1 mentions)
Tritc hyq, by Nikon (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Nikon (1 mentions)
Eclipse 50i, by Nikon (1 mentions)
Nis elements d, by Nikon (1 mentions)
A11073, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 488 antibody, by Thermo Fisher Scientific (1 mentions)
Axiovision fluorescence microscope, by Zeiss (1 mentions)
Hoechst 33342, by Thermo Fisher Scientific (1 mentions)
40 protocols using tmr red in situ cell death detection kit
1
Immunohistochemical Analysis of Pancreatic Beta Cells
2
Nuclei Observation, Mitotic Index, and DNA Fragmentation
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This procedure was used in order to observe nuclei and to determine mitotic index as well as DNA fragmentation. Nuclear architecture and mitotic index was examined with a fluorescent probe Hoechst 33258. Approximately 1000 nuclei were observed in each preparation using a fluorescence microscope (Olympus AX 70, Hamburg, Germany) equipped with broad-spectrum UV excitation. Every morphological change as well as the mitotic index was expressed as a percentage of the total cells. A terminal deoxynucleotidyl transferase (Tdt)-mediated deoxyuridinetriphosphate (dUTP)-nick labelling (TUNEL) assay was performed using a TMR-red in situ cell death detection kit (Roche, Basel, Switzerland), in compliance with the manufacturer’s instructions, as described by Poborilova et al. [46 ]. Approximately 1000 cells per sampling time per treatment were counted in triplicate; the amount of positive BY-2 cells was expressed in percentages.
3
Cell Growth and Apoptosis Assay
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Example 3
To measure the effect of the compounds on cell growth, cells were seeded at 3×104/ml into a 96-well plate. Once attached, cells were treated with various concentrations of AOH1160 for 72 h. Cell growth was measured by the CellTitor-Glo assay (Promega, Madison, Wis.) according to the manufacturer's instructions. To measure apoptosis, cells were seeded at 1×105/ml onto a chamber slide. Once attached, cells were treated with the 500 nM AOH1160 for 24 h. Cells were fixed and analyzed by a TUNEL assay using the TMR red in situ cell death detection kit (Roche Diagnostics, Indianapolis, Ind.).
4
Apoptosis Detection in Cell Samples
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Samples of GSC and RSC (1 ml suspension) were collected at day 0 and 6 and washed three times in phosphate buffered saline (PBS), incubated with fixation solution of 2 % (v/v) paraformaldehyde in PBS for 60 min. Then, samples were treated with permeabilisation solution (0.1 % (w/v) Triton X-100 in 0.1 % (w/v) sodium citrate) and washed three times with PBS. Samples were labelled with TUNEL reaction mixture (TMR-red in situ cell death detection kit, Roche Diagnostics) in darkness at 37 °C for 60 min. Negative (without terminal transferase) and positive (with ethanol treatment at day 0, as described by Hogg et al. [33 (link)]) samples were properly included. For nuclear staining, the samples were washed twice by PBS and stained with 1 μg ml−1 4′,6-diamidino-2-phenylindole (DAPI) for 15 min. Finally, all samples were examined under a Leitz Fluovert fluorescence microscope, with two sets of filters: 360 nm excitation and 420 nm emission for DAPI detection; 540 nm excitation and 620 nm emission for TUNEL, respectively. The percentage of apoptotic TUNEL-positive nuclei was determined by counting at least 200 nuclei.
5
Apoptosis Detection with TMR Red Kit
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According to the manufacturer*s instructions, Apoptosis was detected with the TMR red In Situ Cell Death Detection Kit (Roche, Mannheim, Germany). Terminal transferase was omitted as a negative control. Cells were exposed to DNase I prior to the assay (10 min.; Roche) to provide a positive control. TUNEL-positive cells were counted by an experimenter who was blind to the treatment groups.
6
Immunocytochemistry of Murine Retinal Cryosections
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Mice were sacrificed, eyes enucleated and fixed in 4% paraformaldehyde in PBS for 4 h at 4°C. Eyes were washed in PBS, cryoprotected in 10, 20, and 30% sucrose in PBS, embedded in OCT (VWR), cryosectioned (12 μm), thaw-mounted onto polysine slides (Thermo Fisher Scientific) and stored at −20°C. Serial sections were taken in the optic nerve area. Immunocytochemistry was performed as described before (Palfi et al., 2016 (link)). Sections were incubate with primary antibodies (Table 1 ) overnight at 4°C, then incubated with secondary antibodies conjugated with either FITC, Alexa-Fluor-488, Cy3, and Alexa-Fluor-647 (Jackson ImmunoResearch Laboratories) in 1:400 dilution for 2 h, at RT and nuclei counterstained with DAPI. TUNEL staining was completed using the TMR red in situ cell death detection kit (Roche), as per the manufacturer’s protocol. Subsequent to TUNEL staining, sections were processed for immunocytochemistry. Sections were covered using Hydromount (National Diagnostics).
7
Quantifying Cardiomyocyte Apoptosis
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Isolated adult mouse cardiomyocytes were plated on Lab-TekII Chamber Slide system coated with laminin. Cells were subjected to hypoxia (1 hour) and reoxygenation (2 hours) before staining with 100 nM Nonyl Acridine Orange (Molecular Probes Cat# A1372) in Tyrode's buffer for 30 minutes. Cells were then labeled with TMR red (Roche TMR red in situ cell death detection kit) and imaged by confocal microscopy as previously described.29
8
Apoptosis Detection in Ovarian Follicles
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Follicles were fixed in 4% paraformaldehyde (in 0.1 M sodium phosphate buffer pH 7.4) for 30 minutes, washed twice in PBS, permeabilized (0.3% Triton X-100, 1% BSA, and 1% Sodium citrate in PBS), and washed twice more in PBS before staining. TUNEL staining (TMR Red In Situ Cell Death Detection Kit, Roche, Indianapolis, IN) was used to visualize late-stage apoptosis. Ovaries were transferred to the TUNEL reaction mixture for 2 hours at 37°C then rinsed 3 times in PBS. Positive controls were incubated in DNase I solution (0.1% BSA and 6u/ml DNase I in 50 mM Tris-HCl buffer) for 20 minutes at room temperature prior to staining. Negative controls were incubated in the absence of the enzyme terminal transferase. Follicles were co-stained with DAPI (25 μg/ml, Anaspec, CA) and Alexa Fluor 488 Phalloidin (0.835 μM, Life, NY) in PBS for 1 hour at room temperature. Processed ovaries were mounted in Vectashield (Vector Laboratories, CA) and visualized using a Nikon Eclipse 50i fluorescence microscope and NIS Elements D (Nikon, Melville, NY) with Nikon TRITC HYQ (TUNEL), B-2A (phalloidin) and UV-2E/C (DAPI) filters.
9
Immunofluorescence and In-Situ Hybridization
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Immunofluorescence staining was performed as described previously (Kubin et al., 2011 (link)). Apoptotic cells were detected using the TMR red in situ Cell Death Detection Kit (cat. number 12 156 792 910) from Roche) following the instructions of the manufacturer. For whole mount hybridization E10.5 to E12.5 embryos were hybridized with digoxigenin-labeled Pax3, Myod, Myf5, Lbx1 and myogenin cRNA probes. The hybridized probe was visualized using alkaline phosphatase-coupled anti-digoxigenin antibody and nitroblue tetrazolium-5-bromo-4-chloro-3-indolylphosphate substrate according to manufacturer`s recommendations (Boehringer Mannheim, Mannheim, Germany).
10
In Situ Cell Death Detection
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The TMR red In Situ Cell Death Detection Kit (Roche, Mannheim, Germany) was used in accordance with the manufacturer's protocol. For a positive control, sections were incubated for 15 min at 37 °C in proteinase K, followed by treatment with DNase I (10 U ml−1) for 30 min at 37 °C.
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