Absin 7-Color IHC Kit (Anti-Rabbit and Mouse Secondary Antibody)
Absin 7-Color IHC Kit (Anti-Rabbit and Mouse Secondary Antibody)
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| References | 1. Mingzhu Yang, et al., Chemical-induced chromatin remodeling reprograms mouse ESCs to totipotent-like stem cells. Cell Stem Cell. 2022 Mar 3 2. Xin Guan, et al., Nanoparticle-enhanced radiotherapy synergizes with PD-L1 blockade to limit post-surgical cancer recurrence and metastasis. Nature Communication. 2022 May 20 3. Qian-Yue Zhang, et al., Lymphocyte infiltration and thyrocyte destruction are driven by stromal and immune cell components in Hashimoto's thyroiditis. Nature Communications. 2022 Feb 9 4. Dabbs David J. Diagnostic Immunohistochemistry (M). Beijing: Peking University Medical Press, 2008.9. 5. Beauty ] Ed Harlow, David Lane. Antibody Technical Guidelines (M). Beijing: Science Press, 2002: 79-80, 105. 6. Stack, E. C., et al., Multiplexed immunohistochemistry, imaging, and quantitation: A review, with an assessment of Tyramide signal amplification, multispectral imaging and multiplex analysis. Methods, 2014 70 (1): 46-58. 7. Qian Bangguo. Jiao Lei. Application of multi-label immunofluorescence staining and Doppler imaging in histological study. Chinese Journal of Histochemistry and Cytochemistry, 2017 (4): 373-382. | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| WGK Germany | 0 | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| Usage |
I. Sample requirements 1 , formalin-fixed wax blocks or slides, large pieces of tissue or TMA The sealing wax cannot be obviously damaged. 2 Slide samples, tissues need to be close to slides to avoid wrinkles, slides must not be damaged, scratched or stained. 3 Tissue minimum should contain greater than 1000 Cells. 4 Solid tumor tissues need to be embedded in wax blocks, and necrotic tumor tissues, fine needle punctures and cell slice samples will affect the staining effect. 5 The organization should use 10% Neutral formalin fixation, normal fixation time is 18-24h 。 6 The slice thickness is 3-5μm Left and right, use anti-fall slides. It is recommended that the slides be prepared within one week after fixation. 7 Do not add any adhesive in the water bath fishing process. The sample should be placed on the front and center of the slide. Place the slide vertically on absorbent paper to remove moisture, tap to remove water droplets, do not wipe the slide with paper. 8 The glass slide is placed on 45℃ Place on a hot plate 30min (The duration of natural air-drying slides is not less than 1h ）。 II. Inspection methods 1 、 Required Instruments and Equipment : Pipette, constant temperature drying oven, microwave oven, immunohistochemistry pen, repair cup, dyeing cylinder, timer, incubation wet box, cover glass, fume hood, washing bottle, fluorescence microscope, measuring cylinder 100mL measuring cylinder 1000mL Etc. 2 、 Reagents required: Sterilized deionized water ( abs9259 ), xylene, ethanol ( 100% 、 95% 、 70% ）、 10% Neutral formalin, antigen repair primary antibody, blocking solution, PBST Etc. 3 、 Reagent Preparation: （ 1 ) Dilution of fluorescent dyes: The dye is 200× Mother liquor, use signal amplification reaction solution to press 1:200 Dilute and prepare the dye working solution (use and prepare now, if the signal is weak, the proportion can be reduced to 1:100 ）； DAPI Use sterile water to press 1:100 Dilute the preparation working solution. （ 2 ) Use of secondary antibodies: The kit is a universal secondary antibody for pika. It is not recommended that the sample be homologous to the secondary antibody. Please verify whether the species of the primary antibody matches before the experiment. (Kit secondary antibody is goat species) 4 、 Testing Equipment: A fluorescence microscope or a fluorescence full-film scanning device, TSA Excitation and emission filters suitable for monochromatic fluorescent dyes shall comply with the recommendations in the table. 3. Paraffin section operation steps 1 dewaxing and hydration （ 1 ) Fresh xylene dip tablets 10min , repeat 3 Twice. （ 2 ) Gradient ethanol dip tablets: 100% 5min ； 95% 5min ； 70% 2min 。 （ 3 ) Sterilized water washing tablets 1min , repeat 3 Twice. （ 4 ） 10% Neutral formalin or 4% paraformaldehyde dip tablets 10-30min Sterilized water washing tablets 1min , repeat 3 Twice. (Optional, depending on sample quality) （ 5 ) Dropwise adding membrane breaking agent to permeate 15min ， PBST immersion 3min , repeat 3 Twice. (Optional, you may not do it in general) 2 Microwave repair antigen （ 1 The dewaxed and hydrated glass slide is placed in a repair cup, and the antigen repair solution is used 1× Working fluid immersion. （ 2 ) Place the repair cup in the microwave oven and bring to a high boil. （ 3 ) Low Fire Maintenance 15min (Pay attention to rehydration to prevent excessive evaporation causing dry tablets). （ 4 ) Remove to room temperature and naturally cool to room temperature. 3 Quenching and sealing （ 1 ) Remove that residual lotion from the slide. （ 2 ) Circle the sample area on the slide with a histochemical pen, add peroxidase blocking solution dropwise to cover the sample area, and incubate 10min ， PBST immersion 3min , repeat 3 Twice. （ 3 ) Remove that residual washing solution on the slide, add the blocking solution dropwise, cover the sample area, and incubate at room temperature 60min The blocking solution was removed. 4 Primary antibody incubation （ 1 ) Remove the blocking fluid from the slide. （ 2 ) The diluted primary antibody solution was added dropwise with a pipette to immerse the sample area. （ 3 ) Room temperature moisturizing shaking incubation 1h (need to be optimized for different antibodies) or 4℃ Incubate overnight. （ 4 ) with 1×PBST buffer Immersion slide 3min , repeat 3 Twice. 5 Secondary antibody incubation （ 1 ) Remove any remaining lotion from the slide. （ 2 ) direct dropwise addition HRP Secondary antibody working solution, immerse the sample area. （ 3 ) Moisturizing incubation at room temperature 30min 。 （ 4 ) with 1×PBST buffer Immersion slide 3min , repeat 3 Twice. 6 Fluorescence staining to amplify the signal （ 1 ) Remove any remaining lotion from the slide. （ 2 ) drop-on slide with pipette 1× Dye working solution 50μL-100μL (Use signal amplifier to press 1:200 Dilute) to immerse the sample area. （ 3 ) Room temperature moisturizing shaking incubation 10-15min 。 （ 4 ） 1×PBST buffer Dip slides, room temperature dip slides 3min 。 repeat 3 Twice. 7 A new round of dyeing (single dyeing can be directly performed on the first 8 Step) （ 1 ) After each round of staining, fluorescence microscope can be used to confirm the staining. PBST Cover the sample to prevent dry sheets. （ 2 ) Elution: remove that residual washing solution on the slide, drop the antibody eluent, cover the sample area, 37℃ incubation 20min-30min The eluate was removed. (No Quenching Step Required) （ 3 ) Repeat steps 4-6 （ 4 ) Multiple rounds of staining repeat steps 7 ，（ 1 ） — （ 3 ) Nucleation and sealing were performed after completion 8 Nucleation and sealing Dropwise 1×DAPI Working solution onto the sample, immerse the sample area, and incubate at room temperature 10min 。 use 1×PBST Immersion slide 3 Time, each time 2min 。 Then add antifluorescent quenching sealant dropwise, seal with cover glass, avoid bubbles, long-term storage, please use transparent nail polish to seal the edge of the cover glass. 9 The stained tissue slices were observed and analyzed under a fluorescence microscope. 4. Operation steps of frozen sections (antibody eluate required abs994 ） 1 Quenching, transparency, sealing （ 1 ) Removing the frozen glass slides from the refrigerator and restoring them to room temperature, PBST immersion 3min , repeat 3 Secondary （ 2 ） 10% Neutral formalin or 4% paraformaldehyde dip tablets 10-30min Sterilized water washing tablets 1min , repeat 3 Twice. (Optional, depending on sample quality) （ 3 ) Dropwise adding membrane breaking agent to permeate 15min ， PBST immersion 3min , repeat 3 Twice. (Optional, you may not do it in general) （ 4 ) Circle the sample area on the slide with a histochemical pen, add peroxidase blocking solution dropwise to cover the sample area, and incubate 10min ， PBST immersion 3min , repeat 3 Twice. （ 5 ) Remove that residual washing solution on the slide, add the blocking solution dropwise, cover the sample area, and incubate at room temperature 60min The blocking solution was removed. 2 Primary antibody incubation （ 1 ) Remove the closure on the slide. （ 2 ) The diluted primary antibody solution was added dropwise with a pipette to immerse the sample area. （ 3 ) Room temperature moisturizing shaking incubation 1h (need to be optimized for different antibodies) or 4℃ Incubate overnight. （ 4 ) with 1×PBST buffer Immersion slide 3min , repeat 3 Twice. 3 Secondary antibody incubation （ 1 ) Remove any remaining lotion from the slide. （ 2 ) direct dropwise addition HRP Secondary antibody working solution, immerse the sample area. （ 3 ) Moisturizing incubation at room temperature 30min 。 （ 4 ) with 1×PBST buffer Immersion slide 3min , repeat 3 Twice. 4 Fluorescence staining to amplify the signal （ 1 ) Remove any remaining lotion from the slide. （ 2 ) drop-on slide with pipette 1× Dye working solution 50μL-100μL (Use signal amplifier to press 1:200 Dilute) to immerse the sample area. （ 3 ) Room temperature moisturizing shaking incubation 10min 。 （ 4 ） 1×PBST buffer Dip slides, room temperature dip slides 3min 。 repeat 3 Twice. （ 5 ) dropwise addition of antibody eluate ( abs994 ）， 37℃ incubation 15-20min 。 （ 6 ) Sterilized water washing tablets 1 times, 1×PBST buffer Immersion tablet 2min 。 5 A new round of dyeing (single dyeing can be directly performed on the first 6 Step) （ 1 ) After each round of staining, fluorescence microscope can be used to confirm the staining. PBST Cover the sample to prevent dry sheets. （ 2 ) Repeat steps 2-4 。 （ 3 ) Multiple rounds of staining repeat steps 5 Nucleation and sealing were performed after completion. 6 Nucleation and sealing Dropwise 1×DAPI Working solution onto the sample, immerse the sample area, and incubate at room temperature 10min 。 use 1×PBST Immersion slide 3 Time, each time 2min 。 Then add antifluorescent quenching sealant dropwise, seal with cover glass, avoid bubbles, long-term storage, please use transparent nail polish to seal the edge of the cover glass. 7 The stained tissue slices were observed and analyzed under a fluorescence microscope. V. Description of results:
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| Beilstein | 0 | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| Theory | Multiplex fluorescence immunohistochemical staining is based on the principle of specific binding of antigen and antibody. Secondary antibody labeled with horseradish peroxidase (HRP) is selected to activate the fluorescent dye in the kit and covalently bind the signal to the antigen. In situ multi-target staining of tissues or cells is achieved by multiple rounds of staining cycles with the help of different fluorescent dye labels. | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| Synonym | Multicolor kit | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| Description | There are complex cellular composition in tissue microenvironment, and the phenotype, state, abundance and distribution of these cells have important biological significance and clinical value. It can be presented in situ in tissue with the help of antibody staining. Immunohistochemical staining is a common technique to study tissue morphology and in situ protein expression. Conventional IHC detection can only display a single indicator, and it is difficult to present the cell composition, state and relationship in the complex tissue microenvironment, and this information is crucial for the diagnosis and treatment of diseases! |
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| PubChem CID | 0 | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| Wells | 0 | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| Composition |
TSA Monochromatic fluorescent dye 480 、 TSA Monochromatic fluorescent dye 520 、 TSA Monochromatic fluorescent dye 570 、 TSA Monochromatic fluorescent dye 620 、 TSA Monochromatic fluorescent dye 690 、 TSA Monochromatic fluorescent dye 770 Signal amplification reaction solution, pika universal type HRP Labeled secondary antibody, anti-fluorescence quenching tablet sealing agent, DAPI Peroxidase blocking solution, antibody eluate |
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| General Notes | 1. This kit is only used for immunohistochemistry, not for other purposes. 2. This kit is limited to professionals. 3. Appropriate protective measures should be taken to avoid contact of reagents with skin and eyes. 4. The activity of reagents that have exceeded the expiration date may be reduced, so reagents that have exceeded the expiration date should not be used. 5. If the dyeing components of this kit are mixed with products of other companies, abnormalities may occur during the dyeing process. 6. Dewaxing is not complete, which easily affects the dyeing effect. 7. In order to prevent possible false negative and false positive results, a positive control and a negative control should be carried out simultaneously during the experiment. 8. All kinds of wastes generated in the use of this kit should be treated in accordance with the Regulations on Medical Waste Management. |
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| Storage Temp. | Fluorescent dyes should be kept away from light, stored at 2~8℃, and the validity period is 12 months. | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| Applications | It is mainly used for immunohistochemical staining of tissues, paraffin sections and TMA chips. It can also be used for frozen sections and cell climbing sections, and needs to be matched with abs994 antibody eluate (mIHC specific). | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| Performance Indicator | 1. Conformity: Take the conforming tissue slices (including the positive tissue slices and the negative reagent control). After the corresponding immunohistochemical test, the staining result of the positive control is positive, and the staining result of the negative control is negative. |
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VII. Dye Information Form 
Picture
Picture
Immunohistochemistry
