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IHC Support Pack (Two-Step Method)
Instruction manual
First Edition (Revised on April, 2016)
[PRODUCT INFORMATION]
HRP labeled secondary antibodies combine with primary antibodies on a tissue section to form immune complex. HRP on the antibody can catalyze the reaction of substrate H2O2 with DAB and ultimately to form brown insoluble chromogen. The immunogen is marked in tissue and cells, and the final site is determined by microscopic.
[REAGENTS AND SPECIFICATION]
NO. | Component | Specification | ||||
| 1 | Endogenous peroxidase blocking solution | 3ml | 6ml | 18ml | 55ml | 110ml |
| 2 | HRP labelled secondary antibody（Optional） | 3ml | 6ml | 18ml | 55ml | 110ml |
| 3 | Blocking serum working solution | 3ml | 6ml | 18ml | 55ml | 110ml |
| 4 | Hematoxylin stain | 3ml | 6ml | 18ml | 55ml | 110ml |
| 5 | Sodium citrate antigen repair solution (20×) | 10ml | 20ml | 60ml | 200ml | 400ml |
| 6 | Chromogenic agent (1×) | 3ml | 6ml | 18ml | 55ml | 110ml |
| 7 | DAB solution (50×) | 60ul | 120ul | 360ul | 1.1ml | 2.2ml |
[CANDIDATE INDICATORS]
No. | Name | No. | Name |
IS085-1 | HRP labeled Rabbit anti-human IgG antibody | IS085-9 | HRP labeled Rabbit anti-dog IgG antibody |
IS085-2 | HRP labeled Rabbit anti-mouse IgG antibody | IS085-10 | HRP labeled Rabbit anti-sheep IgG antibody |
IS085-3 | HRP labeled Rabbit anti-rat IgG antibody | IS085-11 | HRP labeled Rabbit anti-cat IgG antibody |
IS085-4 | HRP labeled Rabbit anti-pig IgG antibody | IS085-12 | HRP labeled Rabbit anti-horse IgG antibody |
IS085-5 | HRP labeled Rabbit anti-chicken IgG antibody | IS085-13 | HRP labeled Goat anti-rabbit IgG antibody |
IS085-6 | HRP labeled Rabbit anti-bovine IgG antibody | IS085-14 | HRP labeled Goat anti-mouse IgG antibody |
IS085-7 | HRP labeled Rabbit anti-goat IgG antibody | IS085-15 | HRP labeled guinea pig anti-rabbit IgG antibody |
IS085-8 | HRP labeled Rabbit anti-guinea pig IgG antibody |
[APPLICATION]
This product is only used for immunohistochemistry experiment, suitable for manual and machine mass operation. The reagents only verify for 10% neutral buffered formalin fixed paraffin embedded tissues, and not for others.
[STORAGE AND DEADLINE]
Store at 2~8 degrees, and the shelf life is 6 months.
[EXPERIMENTAL STEPS]
1. Equipments and reagents
a) Pipette, incubator, repair apparatus, immunohistochemistry pen, timer, incubation box, dyeing rack, coverslip, optical microscope, washing bottle.
b) Prepare DAB working solution by diluting DAB solution(50×) with Chromogenic agent.
c) Prepare Sodium citrate antigen repair working solution by diluting Sodium citrate antigen repair solution(20×) with ddH2O.
2. Experimental steps
(1) Baking slice: Mark with a pencil and bake at 65 degrees for 1-2 hours.
(2) Dewaxing: After drying, the paraffin sections are sequentially put in xylene I, xylene II for 30min, 100%, 100%, 95%, 80%, 70% ethanol for 10min, and then put into pure water for 10min.
(3) Antigen repair: high temperature and high pressure method and microwave method.
High temperature and high pressure: sodium citrate antigen repair solution about 1000ml into the pressure cooker. Put the slice into the cooker, cover the pot and preheat to boiling at 1600W. Then close the pressure valve at 1300W, start time when the valve jet. repair time stays 2 minutes.
Microwave method: Put a certain amount of pH 6.0, 0.01M sodium citrate solution into microwave,and heat it to boiling. Then put the slice into the boiling buffer, handle with low fire for 3 minutes. Place it for 8 minutes and then handle with low fire for 3 minutes. Remove water and cool it, take the slice, wash with distilled water for twice, and then wash it with PBS for 3 minutes by 3 times.
(4) Blocking of endogenous peroxidase: Add a proper amount of endogenous peroxidase blocking solution, incubating at room temperature for 10 minutes. And wash with PBS buffer for 3 minutes by 3 times.
(5) The closure of non specific sites: Shake off the residue and cover slices with blocking serum working solution about 200ul, incubating at room temperature for 20min.
(6) Add Primary antibodies: Add an appropriate amount of primary antibodies according to the size of the tissue, incubating for at 37 degrees for 60min. Wash it with PBS buffer for 3 minutes by 3 times.
(7) Select suitable HRP labelled secondary antibody according to species difference, add HRP labelled secondary antibody about 100ml, incubating for 30 minutes at 37 degrees. Wash it with PBS buffer for 3 minutes by 3 times.
(8) Coloration: Add a proper amount of fresh DAB working solution, incubating for 30 seconds-5 minutes at room temperature, and terminate the reaction according to the color under the microscope.
(9) Redyeing: Wash with water, and add hematoxylin stain incubating for 2 minutes. Add 1% hydrochloric acid for 1 second, and wash with PBS to become blue.
(10) Dehydration, transparent, mounting.
(11) Microscopic observation, photographing.
[IMPORTANT NOTES]
1. During dyeing, blank and negative control experiment must be carried out simultaneously.
2. If positive tissue control can not show appropriate positive staining, the results should be invalid.
3. If the HRP labelled secondary antibody is incubated for high temperature or long time, it could lead to excessive dyeing and background staining.
4. Red blood cells and cytochrome C may cause a false positive results.
5. If the dewaxing is incomplete, and it would affect the dyeing. It is suggested that separate the immunohistochemical section dewax from the conventional HE dewaxing.
6. Set up positive and negative control to avoid false positive and false negative results.
7. Over much PBS buffer will dilute reagents, which will reduce dyeing effect. Therefore, remove excess buffer before adding reagents.
8. In the process of operation, please wear gloves, glasses and lab clothes, follow experimental process, and do protective measures.