Rabbit t-PA immunohistochemistry kit
The kit is based on HRP-labeled streptavidin complex (HRP Streptavidin Conjugate, HRP-SA) and can be used to detect specific t-PA antigens in cells and tissues. The kit has high sensitivity, strong specificity, accurate qualitative positioning and clear background. After the t-PA primary antibody used binds to the corresponding target antigen, the biochemical 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 Thing, observe and image under the microscope.
Note: This product is only for laboratory scientific research. This step is an effective step based on our long-term experiments, not necessarily the best method, please adjust and optimize according to the specific scientific research experiments, and welcome customers to discuss with us.
Reagents included in the kit:
Reagent A permeate: 0.1% Triton-X 100 10 mL (optional)
Reagent B Tween 20 5 mL
Reagent C blocking a buffer (for blocking) 20 mL
Reagent D (original imported aliquot) diluted ready-to-use t-PA primary antibody
Reagent E (original imported subpackage) 1 biotinylated goat anti-rabbit IgG
(Concentration 1.5 mg / mL, dilution ratio 1: 300 ~ 1: 500) 50 μL + antibody dilution 20ml
Reagent F 1 HRP-SA complex (concentration 1 μM, dilution ratio 1: 50 ~ 1: 200) 100 μL
Reagent G DAB color developing solution
Reagent F buffered glycerol mountant 10 mL
User-supplied reagents:
1. 10mM TBS (pH7.2 ~ 7.4) trihydroxyaminomethane 1.21g sodium chloride 7.6g plus distilled water 800mL, concentrated hydrochloric acid to adjust the pH value to 7.2 ~ 7.4, and finally set the volume to 1000mL TBS-T: TBS + Tween 20 ( 0.05% by volume)
2. Antigen repair solution (select different repair solutions according to 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 value to 6.0, and finally set the volume To 1000mL or: 0.5M EDTA repair solution (pH8.0) EDTA · 2H2O 186.1g trisodium citrate 2.45g plus distilled water 700mL, adjust the pH value to 8.0 with 10mM NaOH, and finally dilute to 1000Ml paraffin-embedded tissue section for immunostaining
Rabbit t-PA immunohistochemistry kit
Experimental steps (recommended solution):
Paraffin-embedded tissue section 3 ~ 4μm thickness
1. Baked slices: Place the slices on the slice rack and bake at 60 ℃ in a constant temperature oven for at least 1 hr;
2. Dewaxing: Put the slice into a container containing xylene and dewax it 3 times (ie xylene â… , â…¡, â…¢), 10 min each time;
3. Hydration: Slices are hydrated by descending alcohol, absolute ethanol 5min, 95% ethanol 2 times (2min each time), 85% ethanol 2min; 75% ethanol 2min, rinse with tap water, ddH2O wash 2 × 2min;
4. Antigen repair: Antigen repair is carried out according to the recommended method in the antibody manual. High pressure, microwave (temperature up to 98 ~ 100 ℃) or enzyme digestion repair method is often used. The room temperature is naturally cooled, tap water rinses ddH2O for 2 × 2min, TBS (2 × 2min) (see Appendix 1 for the specific repair method) * Note: Some antigens do not need to be repaired, and they go directly to step 5 for sealing.
5. Blocking: add reagent C dropwise and incubate for 30 min in a 37 ° C wet box;
6. Add primary antibody: add reagent D (ready-to-use primary antibody) dropwise, incubate at 37 ℃ in a wet box for 2 hr or 4 ℃ overnight;
7. Washing: TBS-T washing (3 × 5 min);
8. Blocking: add reagent C dropwise and incubate for 10 min in a 37 ° C wet box;
9. Add secondary antibody: add biotinylated secondary antibody (reagent E) diluted with antibody diluent dropwise, and incubate in a 37 ° C wet box for 30 min;
10. Washing: TBS-T washing (3 × 5 min);
11. Blocking: add reagent C dropwise, incubate at 37 ° C in a wet box and block for 20 min;
12. Add HRP-SA: add reagent D diluted with reagent D (1: 50 ~ 200, final concentration 5 ~ 20 nM), incubate in a 37 ° C wet box for 30 min;
13. Washing: TBS-T washing (3 × 5 min), TBS washing (2 × 5 min);
14. Color development: color development using DAB solution (reagent G);
15. Counterstaining: full rinse with tap water, counterstaining, dehydration, and transparency;
16. Mounting: After the tissue specimens are dried, mount with reagent F;
17. Observation and imaging: Observation and imaging under the microscope.
Rabbit t-PA immunohistochemistry kit
Precautions:
1. The buffer solution must be allowed to cool naturally after repair. The slice can be taken out after washing with tap water. The rapid cooling may cause crystallization or antigen blocking.
2. The amount of buffer must ensure that all sections can be soaked, and the used citric acid buffer cannot be used repeatedly.
3. If the reagent is a trace concentrated solution, centrifuge at low speed before use to remove the solution attached to the inner cover and the tube wall to the bottom.
4. Before mounting, be sure to use TBS to wash thoroughly, so as to wash away the residual Tween20 on the tissue, otherwise it will affect the result observation.
5. If the cell nucleus needs to be counterstained, counterstain before mounting or directly mount the tablet containing the nuclear staining reagent.
Attachment 1: Antigen retrieval method
Commonly used antigen repair solution: citrate buffer (0.01M pH6.0), EDTA antigen repair solution (pH8.0 or 9.0) and so on.
1. Enzyme digestion and repair method
The sections were dewaxed and hydrated, washed with TBS, and pepsin or trypsin was added dropwise to the tissues. After incubation at 37 ° C for 20 to 30 minutes, TBS was washed.
2. Microwave antigen repair method
Add the antigen repair solution to the microwave box and microwave to heat it. Place the dewaxed and hydrated slices on a high-temperature plastic slicer and put it in the boiling buffer. Continue microwave for 10 to 15 minutes in the middle or high grade. Take out the microwave box After cooling to room temperature, rinse with tap water and remove the slice. Due to the difference in microwave processing time of different microwave ovens, you must adjust it yourself.
3. Direct high-pressure antigen repair method
Take the repair fluid and boil it in a stainless steel pressure cooker, put the tissue slice on a high-temperature resistant slice rack. After the repair fluid is boiled, put it into the slice rack, cover the lid, and leave the heat source after 1.5 ~ 2.5 minutes after spraying After cooling to room temperature, rinse with tap water and remove the slice. This method is suitable for difficult detection or repair of nuclear antigens.
4. Water-proof high-pressure antigen repair method
Add tap water to the stainless steel pressure cooker to heat it to boiling, add the repair solution to the microwave box and heat it to the boiling point in the microwave oven, place the slices in the microwave box, then put the microwave box in the pressure cooker and cover the lid. Time 4 ~ 8 after jet The heat source can be turned off in min. After cooling naturally to room temperature, rinse with tap water and take out the slice. This method is suitable for difficult detection or repair of nuclear antigens.
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