IHC of MART-1 on Frozen Mohs Melanoma (DAB HRP Brown)
ICH of SOX-10 on Frozen Mohs SCC (DAB HRP Brown)
IHC of CK7 on Frozen Mohs Extramammary Paget’s Disease Tissue (DAB HRP Brown)
|Intended Use||For Mohs In Vitro Diagnostic Use|
|Summary and Explanation||
The Mohs Mouse/Rabbit PolyDetector DAB HRP Brown Detection System is a, 1-step Fab micropolymeric detection system that allows for the demonstration of antigens in cryostat sections, formalin-fixed paraffin-embedded tissue, blood smears, cytosmears, and cell preparations. The Mohs PolyDetector kit has been 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 antigens, in different types of frozen, FFPE tissues and cell preparations.
The increased sensitivity of the Mohs Mouse/Rabbit PolyDetector DAB HRP Brown Detection System allows for rapid staining procedures without compromising stain quality. The Mohs Mouse/Rabbit PolyDetector DAB HRP Brown Detection System is suitable for use with mouse IgG and IgM and rabbit primary antibodies, both monoclonal and polyclonal. The Mohs Mouse/Rabbit PolyDetector DAB HRP Brown Detection System kits are optimized for use with Bio SB TintoFast primary antibodies; however, they are universal kits and therefore should work equally well with antibodies from different vendors, as long as they are properly optimized.
|Presentation||The Mohs Mouse/Rabbit PolyDetector DAB HRP Brown Detection System contains a Peroxidase Blocker solution, an Anti-Mouse/Rabbit Horseradish Peroxidase micropolymer solution, 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 or 10% NBF for 2 minutes at room temperature.
- Rinse with distilled water and air dry the slides for another 2 minutes at room temperature.
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). It is recommended that user choose either a 5 minutes heat-induced epitope retrieval (HIER) method using 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.
- Any of three HIER methods may be used:
a. 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
b. TintoRetriever PT Module or Water Bath Method
Place tissues/slides in a pre-warmed staining dish or coplin jar containing the ImmunoDNA Retriever with Citrate or EDTA at 95°-99° C. Incubate for 10-15 minutes.
c. Conventional Steamer Method
Place tissues/slides in a pre-warmed staining dish or coplin jar containing the ImmunoDNA Retriever with Citrate or EDTA in a steamer, cover and steam for 10-15 minutes.
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 Immunohistochemical Protocol
|Step||Mohs PolyDetector HRP Green or DAB 5 min Protocol||Mohs PolyDetector HRP Green or DAB 10 min Protocol|
|Peroxidase/AP Blocker||0.5 min.||0.5 min.|
|Primary Antibody||2 min||4 min.|
|1st Step Detection||1 min||3 min.|
|Substrate-Chromogen||1 min||2 min.|
|Counterstain / Coverslip||0.5 min||0.5 min.|