| Description | Inquire | Inquire | DescriptionRefer to the product′s Certificate of Analysis for more information on a suitable instrument technique. Contact Technical Service for further support | Product content R669871Component50 TStorageR669871ADNase I1000 U-20℃. Avoid freeze/thaw cycle.R669871B10×Reaction Buffer1mL-20℃. Avoid freeze/thaw cycle. R669871CBuffer DS30 mLRTR669871DBuffer GTL15 mLRTR669871EBuffer GL25 mLRTR669871FProteinase K12.5 mgRTR669871GProteinase K Product content R669871Component50 TStorageR669871ADNase I1000 U-20℃. Avoid freeze/thaw cycle.R669871B10×Reaction Buffer1mL-20℃. Avoid freeze/thaw cycle. R669871CBuffer DS30 mLRTR669871DBuffer GTL15 mLRTR669871EBuffer GL25 mLRTR669871FProteinase K12.5 mgRTR669871GProteinase K Storage Buffer1.25 mLRTR669871HBuffer RW140 mLRTR669871IBuffer RW2 (concentrate)11 mLRTR669871JRNase-Free Water10 mLRTR669871KSpin Columns RS with Collection Tubes50 setsRTR669871LRNase-Free Centrifuge Tubes (1.5 mL)50 EART Product IntroductionThis kit is suitable for effectively purifying total RNA from formalin fixed and paraffin embedded tissues. Suitable for extracting total RNA with improved purity from paraffin embedded tissues or sections less than 30mg. This kit does not require the use of phenol/chloroform extraction or isopropanol precipitation, and can complete the extraction of multiple samples within one hour. This product uses specially optimized lysis solution and protease K to release RNA from formalin fixed or tissue slice samples without overnight operation; After digestion, the sample is incubated at a higher temperature to remove the inhibitory effect caused by formalin cross-linking, effectively releasing RNA from tissue slices and avoiding endangering RNA integrity; The optimized buffer system allows RNA in the lysis solution to specifically bind to the silica gel adsorption membrane, while other pollutants can flow through the membrane; It can be effectively removed through rinsing steps, and the washed RNA can be directly used for experiments such as RT-PCR, Real Time PCR, and Western blot analysis.Self prepared reagents: anhydrous ethanol (newly opened or dedicated for RNA extraction), 10mM PBS (pH 7.4).Preparation and important precautions before the experiment1. Add 0.625ml Protein K Storage Buffer to Protein K to dissolve it and store at -20 ℃. The prepared Protein K should not be left at room temperature for a long time to avoid repeated freeze-thaw cycles, which may affect its activity.2. To prevent RNase pollution, attention should be paid to the following aspects:1) Use RNase free plastic products and gun heads to avoid cross contamination.2) Glassware should be dry baked at a high temperature of 180 ℃ for 4 hours before use, while plastic containers can be soaked in 0.5M NaOH for 10 minutes, thoroughly rinsed with water, and then sterilized under high pressure.3) Prepare the solution using water without RNase.4) Operators should wear disposable masks and gloves, and change gloves frequently during the experiment.3. After obtaining the sample, it should be fixed in 4% -10% formalin as soon as possible, with a suitable fixation time of 14-24 hours. Excessive time can lead to RNA breakage and affect downstream experiments.4. Ensure that the sample before embedding is thoroughly dehydrated, as residual formalin will inhibit the action of Protein K.5. Before the first use, anhydrous ethanol should be added to Buffer RW2 according to the instructions on the reagent bottle label.Before use, please check if there is any crystallization or precipitation in Buffer GTL, Buffer GL, and Buffer DS. If there is any crystallization or precipitation, please dissolve Buffer GTL, Buffer GL, and Buffer DS again in a 56 ℃ water bath.Operation steps1. Sample processing1a. Paraffin embedded sample: Use a surgical knife to trim off excess paraffin from the tissue block, expose the tissue, and cut into 5-10 µ m thin slices.Attention: If the surface of the sample has already been exposed to air, please discard 2-3 pieces that come into contact with the air and do not use them.1b. Samples in fixed solutions such as formalin: Take approximately 20mg of the sample, cut it into small pieces, place it in a centrifuge tube, and add 500 µ 10mM PBS (PH7.4), vortex oscillation, centrifugation at 12000 rpm (~13400 × g) for 1 minute, discard the supernatant, repeat 3 times, and proceed directly to step 3.2. Choose option A or option B to remove paraffinOption AA1. Take approximately 1 × 1cm2 of slices (4-5 slices in total) and place them in a centrifuge tube (prepared by oneself), then add 500 slices µ L Buffer DS, vortex oscillation for 10 seconds. Incubate at 56 ° C for 3 minutes.Centrifuge at A2.12000 rpm for 2 minutes, be careful to discard the supernatant and avoid attracting sediment.Option BB1. Take approximately 4-5 slices of approximately 1 × 1 cm2 and place them in a centrifuge tube (self prepared). Add 1ml of xylene, cover the tube tightly, and vortex for 10 seconds.B2.Centrifuge at 12000 rpm for 2 minutes, be careful to remove the supernatant and avoid removing sediment.B3. Add 1ml of anhydrous ethanol, vortex and shake well. Centrifuge at 12000 rpm for 2 minutes, discard the supernatant, and be careful not to absorb or discard the sediment.B4. Open the tube cover and incubate at room temperature or up to 37 ° C for 10 minutes until there is no ethanol residue.3. Add 150µ L Buffer GTL, resuspended precipitation; Join 10µl Protein K, vortex oscillation mixing.4.Incubate at 56 ℃ for 15 minutes until the sample is completely dissolved. Incubate at 80 ℃ for 15 minutes. Short centrifugation allows the solution on the tube wall to be collected to the bottom of the tube.Note: 1) The purpose of this step is to repair nucleic acids denatured by formaldehyde. Incubating at a high temperature or for too long may cause RNA breakage, resulting in RNA fragments.2) The sample incubated at 56 ℃ can be placed at room temperature until the temperature of the water or dry bath reaches 80 ℃, and then the sample can be incubated at 80 ℃.5. Place on ice for 3 minutes, centrifuge at 12000 rpm for 15 minutes, transfer the supernatant to a new centrifuge tube, be careful not to suck sediment.6. Add 320 to the supernatant µ L Buffer GL, vortex oscillation thoroughly mixed.7. Join 720 µ Mix anhydrous ethanol thoroughly with vortex oscillation.Attention: After adding anhydrous ethanol, there may be a small amount of precipitate precipitation, but it does not affect subsequent operations.8. Add all the solutions obtained in step 7 to the spin columns RS that have been loaded into the collection tube. If the solution cannot be added at once, it can be transferred multiple times. Centrifuge at 12000 rpm for 1 minute, discard the waste liquid in the collection tube, and place the adsorption column back into the collection tube.Optional steps: If genomic DNA needs to be removed, the following steps can be followeda. Add 350 to the adsorption column µ L Buffer RW1, centrifuge at 12000 rpm for 1 minute, discard the waste liquid, and place the adsorption column back into the recovery manifold.b. Preparation of DNase I mixture: Take 52 µ Add 8 RNase Free Water to it µ 10 x Reaction Buffer and 20 µ DNase I (1U/ µ l) Mix well and prepare to a final volume of 80 µ The reaction solution of L.c. Add 80 µ l of DNase I mixture directly to the adsorption column and incubate at 20-30 ℃ for 15 minutes.d. Add 350 to the adsorption column µ L Buffer RW1, centrifuge at 12000 rpm for 1 minute, discard the waste liquid, and place the adsorption column back into the recovery manifold.9. Add 500 to the adsorption column µ Buffer RW2 (check if anhydrous ethanol has been added before use), centrifuge at 12000 rpm for 1 minute, discard the waste liquid in the collection tube, and place the adsorption column back into the collection tube.10. Repeat step 9.Centrifuge at 11.12000 rpm for 2 minutes and discard the waste liquid from the collection tube. Place the adsorption column at room temperature for a few minutes to thoroughly air dry.Note: The purpose of this step is to remove residual ethanol from the adsorption column, which will affect subsequent enzymatic reactions (such as enzyme digestion, PCR, etc.).12. Place the adsorption column in a new RNase free centrifuge tube, and add 20-50µl to the middle of the adsorption column in the air Place RNase Free Water at room temperature for 2-5 minutes, centrifuge at 12000 rpm for 1 minute, collect RNA solution, and store RNA at -20 ℃.Note: 1) The volume of RNase Free Water should not be less than 20 µ l. Small volume affects the recovery rate. 2) If you want to increase RNA production, you can use 20-50 µ Repeat step 12 for the new RNase Free Water.3) If you want to increase the RNA concentration, you can add the obtained solution back to the adsorption column and repeat step 12... Read More | This reagent kit is designed based on the principle that biotin and Streptavidin have a strong affinity. After the primary antibody of rabbit or mouse origin binds to the corresponding target antigen, the biotinylated antibody in this kit • • Rabbit/mouse universal secondary antibody This reagent kit is designed based on the principle that biotin and Streptavidin have a strong affinity. After the primary antibody of rabbit or mouse origin binds to the corresponding target antigen, the biotinylated antibody in this kit • • Rabbit/mouse universal secondary antibody specifically binds to the primary antibody; The biotin labeled on the secondary antibody binds to streptavidin labeled with peroxidase (HRP), forming an antigen-specific primary antibody biotinylated secondary antibody streptavidin complex labeled with HRP. HRP can catalyze substrate colorimetry, thereby inferring the presence and distribution of the tested antigen. The biotinylated secondary antibody and SA-HRP used in this reagent kit all adopt optimized labeling and purification techniques, which make their staining more sensitive and have a lower background. They are suitable for detecting formalin fixed paraffin embedded tissue sections, as well as frozen sections, cell slides, freshly prepared blood smears, etc. The rabbit/mouse universal Streptavidin HRP kit is suitable for use with aladdin ready to use or concentrated antibodies. Composition：Note: This reagent kit is only suitable for IHC experiments where the primary antibody is an immune or mouse derived antibodNotes:1. Add 1 drop (approximately 50) to each slice µ l) Calculation: 3ml can make 60 slices, and 18ml can make 360 slices.2.For tissues with abundant endogenous biotin content, it is best to use endogenous biotin blockers for blocking when using this kit.3. DAB working solution is prepared and used immediately, and the prepared working solution is effective within 1 hour in the dark at 2-8 ° C.4. During the experiment, avoid drying the tissue slices, so the amount of working fluid used during each incubation step must be sufficient to ensure complete coverage of the tissue sample, and incubation should be carried out in a wet box as much as possible.5. To obtain the best experimental results, please make sure to optimize the experimental conditions and reagent dosage.6. DAB is a suspected carcinogen, please take necessary protective measures when using it. 7. This product is only for scientific research and cannot be used for human reactions or treatments.Operation steps：1. Routine processing of samples such as paraffin or frozen tissue sections or cell slides to be tested.1) Preparation for staining of tissue sections or cell slides: a. Dewaxing and hydration of paraffin sections: bake at 60 º C for 1 hour, dewaxing twice with xylene for 5 minutes each time; Then immerse in gradient ethanol (anhydrous ethanol anhydrous ethanol 95% 85% 75% ethanol) and distilled water for 5 minutes each for hydration. b. Frozen sections and cell climbing sections (or climbing sections) were soaked in 0.01 M pH 7.4 PBS and washed 3 times for 5 minutes. Then cover the tissue (or cells) with 0.1% Triton X-100 and infiltrate for 15 minutes. Wash twice with 0.01 M pH 7.4 PBS for 5 minutes.2) Antigen repair of paraffin sections: In most cases, high-pressure repair with citric acid buffer is suitable for paraffin tissue sections. Preparation of repair solution: Add 10 ml of citric acid buffer (IHC antigen repair solution, 100 x) to 1 L of deionized water, and mix well. Repair process: The repair solution is added to a high-pressure cooker, and the repaired slices are immersed in the repair solution (must have no tissue). Cover the pressure cooker cover, heat until evenly sprayed with steam, and start timing from the spraying. After 1-2 minutes, the pressure cooker leaves the heat source and cools naturally to room temperature. Remove the slices, rinse with distilled water, and rinse twice with PBS (0.01 M pH 7.4) for 3 minutes each time.2. Add an appropriate amount of Solution A white solution, which is an endogenous peroxidase blocking solution, and incubate at room temperature for 10 minutes, then rinse thoroughly with PBS.3. Add an appropriate amount of Solution B white solution dropwise, which is sealed with normal sheep serum working solution. Incubate at room temperature for 10 minutes and shake dry.4. Add an appropriate amount of primary antibody working solution (commercial ready to use antibodies or concentrated antibodies diluted in appropriate proportions) dropwise, incubate according to experimental requirements, and then rinse thoroughly with PBS.5. Add an appropriate amount of Solution C yellow solution, namely biotin labeled sheep anti rabbit/mouse secondary antibody working solution, incubate at room temperature for 10 minutes, and rinse thoroughly with PBS.6. Add an appropriate amount of Solution D red solution, which is HRP labeled streptavidin. Incubate at room temperature for 10 minutes and rinse thoroughly with PBS.7. Preparation of DAB color working solution: According to the required amount, mix DAB-A and DAB-B in a volume ratio of 1:19 to obtain DAB color working solution. Alternatively, one drop (approximately 50) can be added per milliliter of reagent B µ l) Reagent A, mix well.8. Color development: Add an appropriate amount of DAB color development working solution to the tissue section or cell slide that needs to be developed, and the color development time is generally 1-5 minutes. Observe and control the color development time under a microscope. When the optimal color development effect is achieved, rinse with tap water to terminate the color development. The colored slices are re stained, dehydrated and transparent, and can be stored for a long time after sealing... Read More |