If the target molecule abundance of fluorescent staining is relatively low or the sample is placed under strong excitation light for a long time, it is easy to have obvious photobleaching effect, that is, fluorescence quenching. Life Technologies has developed ProLong and SlowFade two series of reagents that resist fluorescence signal quenching. The main principle is that it can effectively prevent samples such as fixed cells or tissues after fluorescent staining from being oxidized, thereby preventing the fluorescence signal from being weakened by fluorescence quenching.
The ProLong series of anti-quenching reagents provide several months of protection for the fluorescent signal of the sample. As long as the sample is placed in a dry and protected environment, the fluorescent signal can be not attenuated or quenched for a year or more. However, after processing the sample, the series must be placed in the dark at room temperature for 24 hours before observation and image recording. Otherwise, the fluorescent color will be different from the actual color due to the change of the refractive law (Fig. 1).
The SlowFade series of anti-quenching reagents can be directly observed and image recorded after processing the sample, which ensures that the fluorescence signal does not significantly decrease within 3 to 4 weeks, and can be extended for more time if properly sealed.
The anti-quenching agent is easy to use, just add 1 drop to the slide.
Figure 1. ProLong® Gold increases in refractive index during solidification
Figure 2. ProLong® Gold (Figure A) and SlowFade® Gold (Figure B)
Anti-quenching agent effectively prevents photo-bleaching. Fixing FITC-labeled microspheres with different anti-quenching agents,
100 watts of arc lamp for 30 seconds, 40x/1.3 NA oil sight observation,
12-bit monochrome CCD imaging. The average fluorescence intensity of 20 microspheres was plotted.
Anti-quenching effect of ProLong® Gold and SlowFade® Gold:
(Percentage of initial fluorescence intensity after 30 seconds of continuous illumination with a 40X/1.3 NA objective 100 watt arc lamp)
Steps:
Anti-quenching agents are suitable for most fixed samples, but because they contain glycerin, they are not suitable for certain applications, such as the lipophilic membrane dye Dil.
1.1 Preheating reagent:
Remove the reagent from the refrigerator and equilibrate to room temperature; other heat sources are not recommended, which will reduce the stability of the product.
1.2 Adding the sealing medium (anti-quenching agent):
Remove excess liquid from the section, add 1 drop (or appropriate amount) of anti-quenching agent, cover the coverslip; fix the sample on the coverslip, add 1 drop of anti-quenching agent to the clean slide, carefully cover Place the slide on the place where the quencher is dripped to avoid air bubbles.
1.3 Ready to observe:
When the film is mounted with ProLong, the slide is placed in a dark dry place to be allowed to solidify for several hours to overnight, depending on the thickness of the sample and the relative humidity of the environment. Coagulation is recommended for 24 hours. If long-term storage is required, the edge of the coverslip is sealed after solidification to prevent the shrinkage of the seal to deform the sample. After sealing, the slides are stored vertically in a covered slide box and stored at room temperature, 4 ° C or ≤ -20 ° C, and a desiccant is added. If you need to observe it immediately, fix the corner of the coverslip with nail polish or hot wax to prevent movement and keep the edges clean for easy solidification. After solidification, the edges are sealed for long-term storage.
Mounted with SlowFade, you can observe immediately. For thicker samples, wait until the reagents are completely infiltrated. If necessary, you can also fix the corner of the coverslip with nail polish or hot wax and observe.
Fluorescence microscope settings:
The sample can also be observed before the anti-quenching agent is dried, but the longer the contact between the two, the better the anti-quenching effect, especially ProLong, the refractive index will also change. Reduce exposure time under light with medium-intensity filters that only illuminate when viewing and recording images. Guaranteed good imaging results, with minimal optics, highest digital aperture, low magnification, high quality optical filters, high speed film and efficient lenses.
In addition, ProLong® Gold and SlowFade® Gold anti-quenching agents are also available in DAPI-containing versions, reducing the need for counterstaining DNA.
Related Products
Raw materials for animal medicine
Conventional veterinary drug raw materials are: oxytetracycline hydrochloride, doxycycline, chlortetracycline hydrochloride, lincomycin sulfate, gentamycin sulfate, tylosin tartrate, florfenicol, water-soluble amoxicillin, sulfamethoxazole, enno Floxacin lactate, ciprofloxacin hydrochloride, ciprofloxacin hydrochloride, metronidazole, ribavirin, vitamin C, astragalus polysaccharide, penicillin potassium.
The traditional Chinese medicine extracts in veterinary medicines are derived from natural Chinese herbal medicines.
It is purified, concentrated and purified in a different ratio from the original drug. It not only retains the natural state and biological activity of the original drug composition structure, but also retains the harmless and
The beneficial exogenous substance essence realizes the micro-quantification and standardization of Chinese herbal medicine raw materials, and provides a new way for the breeding industry to realize green production. The following herbal extracts for veterinary use: Anti-pathogenic microorganisms Chinese medicine: Anything that can inhibit or kill the reproduction of pathogenic microorganisms and improve the immunity of the body is called anti-pathogenic microorganisms
Chinese herbal medicine. According to the theory of traditional Chinese medicine, it can be divided into heat-clearing medicines, exopathic medicines, diarrhea and water-repelling medicines, damp-damping and diuretic medicines, phlegm-relieving and cough-relieving medicines, qi-regulating and Xiao-shi pharmacological blood medicines, and phlegm-relieving and anti-asthmatic medicines.
Raw materials for animal medicine,Nicarbazin in Feeds,Nosiheptide Veterinary,Nosiheptide Antibacterial,Human Gastric Mucin
Xi'an Henrikang Biotech Co.,Ltd , https://www.xianhenrikangbio.com