Fluorescein fitc tunel cell apoptosis detection kit

Colon tissues from the indicated mice were paraffin-embedded and sectioned at a thickness of 7 μm. Tissue sections were incubated with dimethyl benzene for 15 min, followed by 85% and 75% ethanol treatments for 5 min. Deparaffinized tissue sections were subjected to antigen rederivation by EDTA buffer, and 3% BSA blockage before incubation with primary antibodies against Claudin1 (Servicebio, GB111032), Occludin (Servicebio, GB111401), Ly-6G (Servicebio, GB11229), and NK1.1 (Biolegend, PK136) overnight at 4 °C and washed with PBS for three times, followed by appropriate secondary antibody (Servcobio, GB21303, GG22404, GB22301) and DAPI (Serviceobio, G1012). Fluorescent Microscopy (Nikon, ECLIPSE C1) was used to identify the indicated protein expression. The TUNEL staining experiment was carried out using the Fluorescein (FITC) TUNEL Cell Apoptosis Detection Kit (Servicebio, G1501). The assay was conducted in accordance with the instructions.

Femurs or tibias were collected from mice and soft tissue was removed. Bone samples were fixed in the 4% PFA for 24 hours and decalcification was performed with 10% EDTA. 4μm-thick paraffin-embedded sections were obtained for further staining. ALP staining was performed by incubating sections with Rabbit polyclonal antibody to Alkaline Phosphatase (1:100, Affinity, Cat. DF6225) overnight at 4˚C. After washing with PBS for 3 times, samples were incubated with HRP conjugated Goat Anti-Rabbit IgG (H + L) (Servicebio, Cat. GB23303) at room temperature for 50 minutes. The slices were photographed under a microscope (Nikon E100, Japan). TRAP staining was performed by using tartrate-resistant acid phosphatase (TRAP) kit (Servicebio, Cat. G1050) according to the manufacturer’s instructions. Imaging was performed on a microscope (Nikon Eclipse E100, Japan). TUNEL staining was performed by using Fluorescein (FITC) TUNEL Cell Apoptosis Detection Kit (Servicebio, Cat. G1501). All kit instructions were followed for paraffin-embedded tissues with a 22-minute proteinase K treatment and fixation with 4% paraformaldehyde. Imaging was performed on a microscope (Nikon, Eclipse C1, Japan).

The liver tissues were dehydrated using graded alcohols, cleared in xylene, and infiltrated with wax. The tissues were then embedded in paraffin using an automatic tissue embedding machine (Leica, Wetzlar, Germany) and placed in a refrigerator at −20 °C for the paraffin wax to solidify. Subsequently, the paraffin blocks were sliced to a thickness of 5–8 μm using a rotary slicer RM2235 (Leica, Wetzlar, Germany) and transferred to slides for drying. For apoptosis detection, the Fluorescein (FITC) TUNEL Cell Apoptosis Detection Kit (Servicebio, Wuhan, China) was used, following the manufacturer’s protocol. The UV excitation wavelength (excitation spectrum, EX) of DAPI was 330–380 nm, emitting blue light (emission spectrum, EM: 420 nm). FITC had an EX of 465–495 nm, emitting red light (EM: 515–555 nm), and the nuclei of positive apoptotic cells appeared red. To preserve fluorescence, the sections were sealed with an anti-fade mounting medium. Fluorescence microscopy was employed to observe the stained tissues, and images were captured for further analysis.

Tumor tissues were fixed and embedded in paraffin. Tissue sections were cut, deparaffinized, repaired with protease K, and permeabilized. Then sections were stained with the Fluorescein (FITC) TUNEL Cell Apoptosis Detection Kit (Servicebio, Wuhan, China). The nuclei were stained with DAPI. Tumor sections were visualized under a fluorescence microscopy (Zeiss, Germany).

In vivo, according to the instructions, renal tissue cell apoptosis was investigated by terminal deoxynucleotidyl transferase‐mediated dUTP nick-end labeling (TUNEL) staining using the DeadEnd™ Fluorometric TUNEL System (Promega, Wisconsin, USA). Detection of positive cells was implemented with by fluorescence microscopy (Carl Zeiss, Jena, Germany) at ×200 magnification. For each sample, 10 images were randomly selected for counting to identify the amount of apoptotic cell nuclei. In vitro, TUNEL staining was carried out using the Fluorescein (FITC) TUNEL Cell Apoptosis Detection Kit (Servicebio, Wuhan, China). The ratio of TUNEL‐positive cells was evaluated in 3 visual fields.
The apoptosis rate of cultured TCMK-1 cells was assessed by an Annexin V-FITC/propidium iodide (PI) kit (4A Biotech, Beijing, China) in combination with flow cytometry (Beckman Coulter, Brea, CA). The preparation of cells was performed following the protocol of manufacturer. In brief, cells were washed twice with PBS and then resuspended in 1×Binding Buffer (100 μl). Afterwards, the cell suspension was added with Annexin V (5 μl) and incubated at room temperature in darkness for 15 min. Lastly, PI (5 μl) and 1× Binding Buffer (400 μl) were inserted and the cells were analyzed by flow cytometry within 1 h.

The TUNEL fluorescence assay was performed according to the instructions of the Fluorescein (FITC) Tunel Cell Apoptosis Detection Kit (Servicebio, Wuhan, China). Details can be found in reference [18 (link)]. Further details are also shown in the Supplementary Materials. The frozen sections of tumor tissue were fixed with 4% paraformaldehyde for about 30–60 min. Then, the fixed sections were washed 2 times with PBS for 10 min each, and PBS added containing 0.1% Triton X-100 and incubated in an ice bath for 2 min. Next, the sections were equilibrated, labeled, nuclear stained, and blocked. Finally, the sections were observed under a fluorescent microscope, and mean fluorescence intensity was quantified by Image J software.