InSituVision Alpha IHC Complete Detection System
InSituVision Alpha IHC Complete Detection System
Product identification
step detection system goat anti-mouse/rabbit HRP, including Blocking serum and DAB chromogen.
Summary and explanation
Step Detection System poly-HRP goat anti-mouse/rabbit IgG Kits with blocking serum and Chromogen (DAB) are highly sensitive and specific polymer-based detection systems. The system is used for the detection of primary antibodies derived from mouse or rabbit. The kits do not contain biotin, so the detection reaction is not affected by endogenous biotin, resulting in less background staining.
Principle of the procedure
Prior to staining, FFPE tissue sections should be processed by deparaffinization and hydration. This is followed by antigen retrieval (HIER or PIER) according to the method indicated in the package insert of the primary antibody. Endogenous peroxidase is inhibited with the included peroxidase blocking reagent. Blocking of nonspecific binding with a protein-blocking reagent is an optional step in the procedure. The use of ready to use primary antibodies is recommended. If antibody concentrates are used, the optimal dilution must be determined in your own system. Polymer-based methods are used for signal amplification. The secondary antibody is conjugated to the enzyme HRP using a synthetic polymer support. This approach increases the number of available enzymes or ligands, resulting in a higher turnover rate of the chromogen. Enzymatic activation of the chromogen results in a visible reaction product at the antigen site. Application of the attached and freshly prepared DAB reagent results in a brown product. Each step is incubated for a precise time and requires intermediate wash steps with recommended wash buffer. The final step is counterstaining, which yields a blue nuclear stain. This is followed by mounting with a mounting medium. For information on product
recommendations, please refer to the section “Additional material required but not supplied”. The results are interpreted under the light microscope
Staining procedure
The following data are recommendations. Due to differences in tissue fixation and processing, as well as general characteristics of the laboratory equipment used and prevailing laboratory conditions, it may be necessary to adjust incubation times. The best possible procedure will be determined and verified by the user.
If using an autostainer, carefully read the instruction manual before use. The recommended reagent volume is 200 μl per slide.
| 1. Deparaffinization and rehydration of the tissue sections on the slides. |
| 2. Wash in IHC wash buffer 2 x 5 min. |
| 3. Enzyme digestion or heat pretreatment can be performed if required (see primary antibody data sheet).
A suggested HIER protocol: Preheat Antigen Enhancer (HIER buffer) to 95 – 99 °C and incubate sections in it for 20 min. Cool sections in hot Antigen Enhancer to RT for 20 min. Wash sections in IHC wash buffer for 2 x 5 min, drain off liquid. |
| 4. Blocking of endogenous peroxidase with the included peroxidase blocking reagent (optional, material not provided) 5 min |
| 5. Wash in IHC wash buffer 2 x 5 min |
| 6. Incubate sections in blocking serum 30min |
| 7. Wash in IHC wash buffer 2 x 5 min |
| 8. Primary Antibody 60 min
Apply enough volume to completely cover the tissue section. Incubate in humidity chamber at RT. Apply sufficient volume to completely cover the tissue section. |
| 9. Wash in IHC wash buffer 2 x 5 min |
| 10. Apply Polymer-HRP goat anti-mouse/rabbit igG
30 min Apply enough volume to completely cover the tissue section. |
| 11. Wash in IHC wash buffer 2 x 5 min |
| 12. Chromogen: DAB 2 – 10 min
Prepare and incubate the solution according to the working instructions of the chromogen supplied. Apply enough volume to completely cover the tissue section. |
| 13. Wash with Aqua Dest. 2 x 2 min |
| 14. Manual counterstaining with hematoxylin
1 min |
| 15. Rinse well with Aqua Dest. |
| 16. Mounting with suitable mounting media. |
| 17. Evaluation of the sections under the light microscope. |
Interpretation of results
At the end of the procedure, there is a colored reaction product at the antigen site localized by the primary antibody.
DAB results in light-brown reaction products.
Chromogen-specific color should appear in the positive control at the expected sites. Nonspecific staining can be recognized by the fact that it appears as rather diffuse on the slides treated with the negative control reagent. The nuclei are stained blue by the hematoxylin counterstain.
Positive and negative controls are initially evaluated by a qualified pathologist. If the control slides are suitable, evaluation of the patient samples can begin. The intensity of the positive staining must be evaluated in light of the background staining of the negative reagent
control.
Note: A negative result means that the antigen in question was not detected, but not that the antigen is not present in the cells/tissues tested. An antibody panel may be used to support the results in some circumstances. In addition, the morphology of all tissue samples should be examined using a hematoxylin/eosin-stained section. Interpretation of the morphologic findings of the patient specimens as well as the clinical data should only be performed by a qualified pathologist.
Warnings and precautions
- Use only by trained and qualified personnel.
- If used as directed, the product is not classified as a hazardous substance, so that no health hazard is to be expected. The safety data sheet is available on request.
- As with all products of biological origin, these must be handled appropriately.
- Do not use the reagents after the expiry date.
- Take appropriate precautions when handling reagents. Wear appropriate protective clothing when working.
- Manage waste disposal in accordance with local, state, and federal standards. Materials of human or animal origin must be handled as biohazardous materials and disposed of with proper precautions.
- Avoid microbial contamination of reagents, as this may cause erroneous results.
Materials provided
|
Tube Label |
Volume
(15ml pack) |
Volume
(50ml pack) |
| (1) Blocking Serum | 20 | 60 |
| (2) Poly-HRP goat anti mouse/rabbit IgG | 15 | 50 |
| (3) DAB Substrate | 20 | 60 |
| (3A) Reagent A, DAB Chromogen (20X concentrated) | 1 | 3 |
| (3B) Reagent B: DAB Chromogen Enhancer (20X concentrated) | 1 | 3 |
The batch number is indicated on the product label.
Reagents are ready to use and have been optimized for staining. No further dilution, reconstitution, mixing or titration is required.
For DAB chromogen reagents (3, 3A, 3B) please see reagent preparation section.
Materials required but not provided
The following materials may be required for staining with the polymer system, but are not supplied.
– Positive and negative controls
– Slides (positively charged) and coverslips
– Water bath with accurate temperature recording
– humidity chamber
– Staining jars
– Stopwatch
– Xylene or xylene substitute
– Ethanol
– Deionized or distilled water
– Primary antibody, ready to use or concentrate
– Antibody diluent solution
– Antigen retrieval reagent, e.g. InSituChem® Antigen Retrieval Buffer, Cat. No. D004
– Hematoxylin
– Mounting medium
– Wash buffer, e.g. InSituChem® IHC Wash Buffer, Cat. No. D003
– Tap water/bluing reagent (e.g. ammonia water)
– Light microscope
Storage and handling
Upon receipt in the laboratory. transfer reagent 3A to -20°C freezer.
Store other kit components at 2-8°C.
When stored correctly, the product is stable until the expiration date printed on the vial. Do not use the reagent after the expiration date.
To maintain proper reagent delivery and product stability, replace the cap after each use and immediately refrigerate the vial in an upright position.
Date of publication or revision
2022-05-08
Change(s) made: –
Explanation of symbols
