Rat VEGF Immunohistochemistry Kit Instructions Rat VEGF Immunohistochemistry Kit This kit is based on HRP Streptavidin Conjugate (HRP-SA) and can be used to detect cells and tissues. Specific VEGF antigen. The kit has high sensitivity, specificity, accurate positioning and clear background. After the VEGF primary antibody used binds to the corresponding target antigen, the biological secondary antibody specifically binds to the primary antibody, and finally HRP-SA is added to form an antigen-specific primary antibody-biotinylated secondary antibody-HRP-SA complex. The imaging was observed under a microscope. Reagents included in the kit: Reagent A Permeate: 0.1% Triton-X 100 10 mL (optional) Reagent B Blocking Buffer (for blocking) 20 mL Reagent C (original dispensing) diluted ready-to-use VEGF Anti-(2.5ml) Reagent D (original imported package) Biotinylated goat anti-rabbit IgG 1 (concentration 1.5 mg/mL, dilution ratio 1:300~1:500) 50 μL+ antibody dilution 20 ml reagent E HRP- SA complex 1 (concentration 1 μM, dilution ratio 1:50~1:200) 100 μL reagent F DAB coloring solution 5ml user-supplied reagent: 1. 10mM TBS (pH 7.2~7.4) Trihydroxymethane 1.21g sodium chloride 7.6g plus distilled water 800mL, concentrated hydrochloric acid adjusted to pH 7.2~7.4, finally fixed to 1000mLTBS-T:TBS+Tween 20 (0.05% by volume Than) 2. Antigen repair solution (select different repair solutions depending on the detection antigen) 10mM pH6.0 Citric acid buffer citric acid 0.38g Trisodium citrate 2.45g plus distilled water 900mL, concentrated hydrochloric acid to adjust the pH to 6.0, and finally to 1000mL Or: 0.5M EDTA repair solution (pH 8.0) EDTA·2H2O 186.1g trisodium citrate 2.45g plus distilled water 700mL, adjust the pH to 8.0 with 10mM NaOH, and finally set the volume to 1000Ml3. Buffer glycerin mounting agent 10 mL4. Tween 20 5 mL paraffin-embedded tissue section immunostaining experimental procedure (recommended): paraffin-embedded tissue section 3~4μm thickness 1. baking sheet: Place the slice to be sliced ​​on a slice rack and bake at least in a constant temperature oven at 60 °C 1 hr; 2. Dewaxing: Slices were placed in a container containing xylene for 3 times (ie, xylene I, II, III) for 10 min each time; 3. Hydration: The slices were hydrated by descending alcohol. Anhydrous ethanol for 5 min, 95% ethanol for 2 times (2 min each), 85% ethanol for 2 min; 75% ethanol for 2 min, tap water rinse, ddH2O wash for 2×2 min; 4. Antigen retrieval: antigen retrieval according to the recommended method of antibody specification, Often use high pressure, microwave (temperature reaches 98 ~ 100 ° C) or enzyme digestion repair Method, natural cooling at room temperature, tap water, ddH20 washed 2 × 2min, TBS and washed (2 × 2min) (specifically see enclosure repairing method 1) * Note: Some antigens Needless repair, go directly to step 5 is closed. 5. Blocking: Add reagent B, incubate in a wet box at 37 °C for 30 min; 6. Add primary antibody: add reagent C (ready-type primary antibody), incubate at 37 °C for 2 hr or 4 °C overnight; Washing: TBS-T washing (3 × 5 min); 8. Closure: Add reagent B, incubate in a wet box at 37 ° C for 10 min; 9. Add secondary antibody: Add biotinylated secondary antibody diluted with antibody dilution ( Reagent D), incubate in a wet box at 37 ° C for 30 min; 10. Wash: TBS-T wash (3 × 5 min); 11. Block: add Tween 20, 37 ° C wet box incubation for 20 min; HRP-SA: Dilute reagent E (1:50~200, final concentration 5~20 nM) diluted with antibody dilution, incubate for 30 min in 37 °C wet box; 13. Wash: TBS-T wash (3×5 Min), TBS washing (2 × 5 min); 14. Color development: application of DAB solution (reagent F) color development; 15. counterstaining: tap water washing, counterstaining, dehydration, transparent; 16. sealing: to be organized After the specimen is dried, the reagent is buffered with a glycerin mounting medium; 17. Observation imaging: Imaging is observed under a microscope. Note: 1. After the repair, the buffer should be naturally cooled. After washing with tap water, the slice can be taken out. The quenching may lead to crystallization or antigen blocking. 2. The amount of buffer must be such that all sections can be soaked, and the used citrate buffer cannot be used repeatedly. 3. If the reagent is a micro-concentrate, centrifuge at low speed before use to remove the solution attached to the inner cap and the tube wall to the bottom. 4. Always use TBS to fully wash before sealing to wash away the residual Tween 20 on the tissue, otherwise it will affect the result observation. 5. If the nuclei need to be counterstained, re-stain before the seal or directly seal with a seal containing the nucleating agent. Attachment 1: Antigen repair method commonly used antigen repair solution: citrate buffer (0.01M pH6.0), EDTA antigen repair solution (pH 8.0 or 9.0) and so on. 1. Enzymatic digestion and repair method, dewaxing and hydration treatment, TBS washing, adding pepsin or trypsin to the tissue, incubating at 37 °C for 20~30min, then TBS can be washed. Second, the microwave antigen repair method in the microwave box to add the antigen repair liquid microwave heating to boiling, the dewaxed hydrated slices placed on the high temperature plastic slice rack, placed in the boiling buffer, medium or high-grade continuous microwave 10 ~15min, remove the microwave box and cool to room temperature, rinse with tap water, and take out the slices. Due to the difference in microwave processing time of different microwave ovens, they must be adjusted by themselves. 3. Direct high-pressure antigen retrieval method Take the repair liquid and heat it to boiling in a stainless steel pressure cooker. Place the tissue section on the high-temperature-resistant slice rack. After the repair liquid boils, put it into the slice holder and cover the lid. After the jet is pressed, the timing is 1.5~2.5. Min can be separated from the heat source, naturally cooled to room temperature, rinsed with tap water, and the slices are taken out. This method is suitable for the detection of more difficult or nuclear antigens. Fourth, water-proof high-pressure antigen repair method Add stainless steel pressure cooker to the boiling water in the stainless steel pressure cooker, add the repair liquid in the microwave box to the boiling in the microwave oven, put the slice into the microwave box, then put the microwave box into the pressure cooker and cover the pot Cover, after 4~8 min after the jet, turn off the heat source, naturally cool to room temperature, rinse with tap water, and take out the slice. This method is suitable for the detection of more difficult or nuclear antigens.
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