SPECIFICATION
This kit provides a classic, fast, and simple method for detecting cell apoptosis. When cells undergo apoptosis, chromatin undergoes pyknosis. So after staining with Hoechst 33258, under a fluorescence microscope, the nuclei of normal cells appear normal blue, while those of apoptotic cells appear dense or fragmented, with some turning white in color. The typical Hoechst 33258 stained cell image is shown in the following figure. This kit provides fixation solution, staining solution, and anti fluorescence quenching sealing solution. Suitable for adherent cells, suspension cells, and tissue sections. The entire process of cell apoptosis detection can be completed in just 25 minutes.
Storage: 4 ℃, protected from light, valid for 6 months.
Precautions:
1.A fluorescence microscope or laser confocal microscope that can observe blue fluorescence is required.
PBS or 0.9% NaCl solution is required.You need to provide your own cover glass and slide.
2.Fluorescent substances are prone to quenching, and dyed samples should be stored away from light.
When using anti quenching sealing liquid, quenching can be slowed down, but it is still advisable to avoid light as much as possible.
3.This product is limited to scientific research by professionals and cannot be used for clinical diagnosis or treatment.
For your safety and health, please wear laboratory clothes and disposable gloves for operation.
Usage:
Adherent cell
1. Soak the clean cover glass in 70% ethanol for 5 minutes or more, blow dry it in a sterile ultra clean table or wash it three times with sterile PBS or 0.9% NaCl solution, and then wash it once with cell culture medium. Place the cover glass in a six well plate and plant it into the cell culture overnight until it is about 50% to 80% full.
After stimulating cell apoptosis, suck out the culture medium and add 0.5mL of fixative for 10 minutes or more (it can be overnight at 4 ℃).
2. Remove the fixing solution, wash twice with PBS or 0.9% NaCl, each time for 3 minutes, and absorb all the liquid. When washing, it is advisable to use a rocking bed or manually shake it.
Add 0.5mL Hoechst 33258 staining solution and stain for 5 minutes. It is also advisable to use a shaking table or manually shake it several times.
3. Remove the staining solution, wash twice with PBS or 0.9% NaCl, each time for 3 minutes, and absorb all the liquid. When washing, it is advisable to use a rocking bed or manually shake it. Drop a drop of anti fluorescence quenching sealing solution onto the slide, cover it with a cover glass with cells, and let the cells come into contact with the sealing solution, avoiding bubbles as much as possible.
4. Fluorescence microscopy can detect blue nuclei. The excitation wavelength is around 350nm, and the emission wavelength is around 460nm. Please refer to the figure below for the graph.
Suspended cell
1. Centrifuge and collect cell samples into a 1.5mL centrifuge tube, add 0.5mL of fixative, slowly suspend the cells, and fix for 10 minutes or more (can be overnight at 4 ℃).
2. Centrifuge to remove the fixed solution and wash twice with PBS or 0.9% NaCl for 3 minutes each time. Manually shake several times during washing.
3. After centrifugation, absorb most of the liquid and retain about 50% μ L liquid, then slowly suspend the cells and add them dropwise to the slide, trying to distribute the cells evenly. Dry slightly to prevent the cells from sticking to the slide and making it difficult for the liquid to flow.
Evenly drop 0.5mL Hoechst 33258 staining solution and stain for 5 minutes. Use absorbent paper to remove the liquid from the edge and let it dry slightly.
4. Remove the staining solution, wash twice with PBS or 0.9% NaCl, each time for 3 minutes, and absorb all the liquid. When washing, it is advisable to use a rocking bed or manually shake it.
5. Drop a drop of anti fluorescence quenching sealing solution onto the slide and cover it with a clean cover glass to avoid bubbles as much as possible. Fluorescence microscopy can detect blue nuclei. The excitation wavelength is around 350nm, and the emission wavelength is around 460nm. Please refer to the figure below for the graph.
Tissue slicing
1. After routine embedding of slices, according to the different types of slices, they can be processed to be used for immunohistochemical staining.
Wash twice with PBS or 0.9% NaCl for 3 minutes each time, and absorb all liquid. When washing, it is advisable to use a rocking bed or manually shake it. It can be operated in a six hole plate.
2. Add 0.5mL Hoechst 33258 staining solution and stain for 5 minutes. It is also advisable to use a shaking table or manually shake it several times.
3. Remove the staining solution, wash twice with PBS or 0.9% NaCl, each time for 3 minutes, and absorb all the liquid. When washing, it is advisable to use a rocking bed or manually shake it.
4. Carefully place the slice on a glass slide, drop a drop of anti quenching sealing solution, and cover it with a clean cover glass to avoid bubbles as much as possible.
5. Fluorescence microscopy can detect blue nuclei. The excitation wavelength is around 350nm, and the emission wavelength is around 460nm. Please refer to the figure below for the graph.