Product Information
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Product Name
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Cat. No.
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Spec.
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S-Vision IHC Kit (Poly-HRP Conjugated Goat Anti-Mouse and Rabbit lgG (H+L))
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AO-03-G1313-100T
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100T
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Product Description
This product utilize a dextran backbone to which multiple HRP enzyme and antibodies molecules are conjugated. This polymer creates a highly sensitive, readily available and one-step non-biotin detection system for immunohistochemical and immunocytochemical staining.This system avoids the use of streptavidin and biotin, and therefore eliminates non-specific staining as a result of endogenous biotin..It has a wide adaptability to sample processing, reducing the variability caused by different tissue processing methods.The reagent is ready-to-use, which does not need to be diluted to obtain the best staining ratio, reducing experimental operation errors.Using 100 μL secondary antibody and DAB working solution per sample, the kit can test 100 samples in total.
Storage and Shipping Conditions
Wet ice packs for transportation, store at 2-8℃ for 12 months.
Product Content
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Component Number
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Component
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AO-03-G1313-100T
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G1313-1
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S-vision poly-HRP conjugated Goat Anti-Mouse and Rabbit lgG(H+L), Ready to use
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10 mL
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G1313-2
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50×DAB Stock Solution
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250 μL
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G1313-3
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DAB Diluent Solution
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12.5 mL
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Product Manual
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One copy
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The Staining Process
1. Manually IHC Staining
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Procedures
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Reagent
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Protocol
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Dewax
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BioDewax and Clear Solution (Recommendation G1128)
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The sections were placed in BioDewax and Clear SolutionⅠ10min-BioDewax and Clear Solution Ⅱ10min-BioDewax and Clear Solution Ⅲ10min-absolute ethanolⅠ5min-absolute ethanolⅡ5min-absolute ethanol Ⅲ 5min-wash in pure water.
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Antigen Retrieval
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Citrate Antigen Retrieval Solution pH6.0 (Recommendation G1202)or EDTA Antigen Retrieval Solution pH 9.0 (Recommendation G1203)
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Microwave Heating: Heating to boiling, maintain a temperature above 98℃ for 15-20 min, and cooling to room temperature;
Pressure cooker heating: Heat in the pressure cooker to air injection for 2 min;
Note: The repair time is also related to the fixed time and the antigen epitope, so the conditions can be explored.
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Blocking of the endogenous peroxidases
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3% Hydrogen peroxide
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Dipping or incubate at room temperature for 10 min. Wash the slide in PBS (pH 7.4) on a shaker 3 times for 5 min each time.
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Blocking
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BSA (Recommendation GC305010)
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The 3% BSA working solution was prepared, and the tissue was completely covered by drops, and incubated at room temperature for 30 min.
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Primary antibody
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commercialization Primary antibody
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Add a working solution to completely cover the tissue, usually 100 μL, and incubate at 4℃ overnight or 37℃ for 2 hours.
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Secondary antibody
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S-vision poly-HRP conjugated antibody
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Drop this product until tissue is completely covered, usually 100 μL, incubated at room temperature for 20 min.
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DAB staining
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DAB Chromogenic Kit (Recommendation G1212)
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Add the prepared DAB working solution to completely cover the tissue and develop color for 5-10 min.
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Hematoxylin staining
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Hematoxylin Solution (Recommendation G1004)
hematoxylin differentiation solution (Recommendation G1039)
hematoxylin bluing solution (Recommendation G1040)
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The sections were directly stained into hematoxylin staining solution for 3-5 min and washed with water; then stained by hematoxylin differentiation solution for 2-5 s and washed with water; hematoxylin bluing solution for1min and washed with water.
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Dehydration and Mounting
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Mounting Medium
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75% alcohol 5min-85% alcohol 5min-absolute ethanolⅠ5min-absolute ethanolⅡ5min- n-butyl alcohol 5min-xylene Ⅰ5min, The section were removed from xylene and mounting.
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2. Automation in IHC
This protocol uses the Servicebio IHC-48 histochemical instrument for reference only. For other instruments, refer to the instructions for each instrument.。
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Procedures
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Reagent
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Protocol
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Dewax
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BioDewax and Clear Solution (Recommendation G1128)
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Dewax at 62℃for 3min, repeated three times, Wash with absolute ethanol for 3 times and wash twice with pure water.
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Antigen Retrieval
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Antigen Retrieval Solution(For histochemical instrument)
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Antigen Retrieval Solution (For histochemical instrument), Heat at 100℃ for 25 min and cool for 20 min.
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Blocking of the endogenous peroxidases
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3% Hydrogen peroxide
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Incubate at room temperature for 10 min.
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Blocking
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BSA (Recommendation GC305010)
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The 3% BSA working solution was prepared, and incubated at room temperature for 10 min.
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Primary antibody
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commercialization Primary antibody
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Working solution was incubated at 37℃ for 15 – 30 min and washed three times with PBS.
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Secondary antibody
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S-vision poly-HRP conjugated antibody
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incubated at RT for 20 min and washed three times with PBS.
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DAB staining
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DAB Substrate Kit (Recommendation G1212)
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Add the prepared DAB working solution and develop color for 5-10 min, washed three times with water.
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Hematoxylin staining
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Mayer Hematoxylin Solution
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Incubate at room temperature for 10 min., washed three times with water.
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Dehydration and Mounting
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Mounting Medium
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75% alcohol 5min-85% alcohol 5min-absolute ethanolⅠ5min-absolute ethanolⅡ5min- n-butyl alcohol 5min-xylene Ⅰ5min, The section were removed from xylene and mounting.
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Note
1. Reagent 1 is a ready-to-use without dilution.Reagents 2 and 3 are diluted in a 1:50 volume ratio.
2. If the background is deep, the primary antibody can be diluted by gradient to obtain better results.
3. The quantity of this kit is calculated according to the manual staining method. If use in the histochemical instrument, you need to calculate the dosage ratio of each component according to the steps of the instrument or directly purchase the special reagent set for the histochemical instrument..
4. Wear protective equipment. for your safety
For Research Use Only!
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