IHC of Mohs PolyDetector Plus HRP Green Detection System staining TintoFast SOX-10 on Frozen Mohs Melanoma
IHC of Mohs PolyDetector HRP Green Detection System staining TintoFast Ki-67 on Frozen Squamous Cell Carcinoma Tissue
IHC of Mohs PolyDetector Plus HRP Green Detection System staining TintoFast EpCAM BerEP4 on Frozen Mohs SCC
|Intended Use||For Mohs In Vitro Diagnostic Use|
|Summary and Explanation|
The Mohs Mouse/Rabbit PolyDetector Plus DAB HRP Green Detection System is a non-biotin 2-step Fab micropolymeric detection system that allows for the demonstration of antigens in cryostat sections, formalin-fixed paraffin-embedded tissues, blood smears, cytosmears, and cell preparations. The technology was developed using a biopolymer conjugated to monomeric Fab’ immunoglobulin fractions targeting the Fc region of Mouse and Rabbit antibodies. Additionally, the biopolymer is labeled with high quality HRP for maximum sensitivity. This ensures excellent cellular penetration which generates consistent, reproducible sensitive and specific immunostainings for all types of nuclear, cytoplasmic and membranous localization in frozen and FFPE tissues.
The increased sensitivity of the Mohs Mouse/Rabbit PolyDetector Plus DAB HRP Green Detection System allows for rapid staining procedures without compromising stain quality, and is suitable for use with mouse IgG and IgM and rabbit primary antibodies, both monoclonal and polyclonal. The Mohs Mouse/Rabbit PolyDetector Plus DAB HRP Green Detection System kits are optimized for use with the Bio SB TintoFast primary antibodies and the Bio SB TintoDetector ImmunoDNA System; however, they are universal kits and therefore should work equally well with antibodies from different vendorsas long as they are properly optimized.
|Presentation||The Mohs Mouse/Rabbit PolyDetector Plus DAB HRP Green Detection System contains Anti-Mouse/Rabbit Horseradish Peroxidase micropolymer link and label,a DAB Buffer Substrate, and a DAB Chromogen solution. All components are buffered with stabilizers and a non-azide anti-microbial agent.|
Mohs IHC Procedure
Specimen Preparation of Mohs Frozen Tissues
- Embed the specimen in OCT inside a cryostat.
- Cut sections at 4-5 µm and mount on a positively charged glass slide such as the Bio SB Hydrophilic Plus Slides (BSB 7028).
- Air dry the slide at room temperature for 2 minutes and then incubate the slide at 60 °C for 3 minutes in an incubator or dry bath.
- Fix in 100% acetone and air dry or fix with 10% NBF for 2 minutes at room temperature then rinse with distilled water and air dry.
Tissue Pretreatment Procedure for Mohs Frozen Tissues
- Subject tissues to epitope retrieval using Bio SB ImmunoDNA Retriever with Citrate (BSB 0020-BSB 0023), ImmunoDNA Retriever with EDTA (BSB 0030-BSB 0033) or Mohs ImmunoDigestor (BSB 0324- 0326) based on antibody pretreatment recommendations. It is recommended that an user choose either a 3-5 minutes heat-induced epitope retrieval (HIER) method using a TintoDetector ImmunoDNA System (BSB 7000) or a pressure cooker (BSB TintoRetriever Pressure Cooker) at 110 °C, or a 1-minute proteolytic induced epitope retrieval (PIER) method (for Cytokeratins using 10% NBF fixed tissues). Morphology is better with 10% NBF fixed tissues, especially when using proteolytic induced epitope retrieval method, however, antigen maybe easier to detect on the 100% acetone fixed tissue.
- Two diffrent HIER methods may be used:
a. TintoDetector ImmunoDNA System
Pair theTintoDetector Cap Gap Plus slides (BSB 7006) with Mohs Frozen Tissues face to face and dip slides into a retrieving solution. Make sure the retrieving solution covers the tissues. Insert the TintoDetector Slide handle with the slides inside the TintoDetector incubatorat110° Cand incubate the slides for 3-5 minutes.
b. TintoRetriever Pressure Cooker or Equivalent
Place tissues/slides in a staining dish or coplin jar containing the ImmunoDNA Retriever with Citrate or EDTA, and place on trivet or staining dish support in the pressure cooker. Add 1-2 inches of distilled water to the pressure cooker and turn heat to high (110-121° C). Incubate for 5 minutes. Open and immediately transfer slides to room temperature. Cool off for 5 -10 min.
3. For PIER, apply Mohs ImmunoDigestor (BSB 0324- 0326) at room temperature for 1 min then wash.
IHC Detection Procedure
- After HIER or PIER, transfer slides to ImmunoDNA washer and let stand for 1-2 minutes.
- For manual staining, perform antibody incubation at ambient temperature. For automated staining methods, perform antibody incubation according to instrument manufacturer’s instructions.
- Wash slides with ImmunoDNA washer or DI water.
- Continue IHC detection protocol. Wash slides between each step with ImmunoDNA washer solution.
Abbreviated Mohs PolyDetector Plus Immunohistochemical Protocol
|Step||Mohs PolyDetector Plus DAB 20 min Protocol||Mohs PolyDetector Plus HRP Green 20 min Protocol|
|HIER||3 – 5 min.||3 – 5 min.|
|Primary Antibody||5 min.||5 min.|
|1st Step Detection||4 min.||4 min.|
|2nd Step Detection||4 min.||4 min.|
|Substrate-Chromogen||2 min.||2 min.|
|Counterstain / Coverslip||0.5 min – 1 min.||0.5 min – 1 min.|