TintoFast CK 5 & 6 (D5 & 16B4), MMab

Anti-CK 5 & 6 is a cocktail of mouse monoclonal antibodies derived from cell culture supernatant that is concentrated, dialyzed, filter sterilized and diluted in buffer pH 7.5, containing BSA and sodium azide as a preservative.

Mohs IHC Procedure

Specimen Preparation of Mohs Frozen Tissues

1. Embed the specimen in OCT inside a cryostat.
2. Cut sections at 4-5 µm and mount on a positively charged glass slide such as the Bio SB Hydrophilic Plus Slides (BSB 7028) or in the lower third of the TintoDetector Cap Gap slides (BSB 7006).
3. 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.
4. Fix in 100% acetone or 10% NBF for 2 minutes at room temperature, Using 10% NBF produces better morphology if using Mohs ImmunoDigestor.
5. For slides fixed in avetone, let it air dry. For slides fixed in NBF, rinse with distilled water and air dry.

Pretreatment of Mohs Frozen Tissues

1. Transfer slides to ImmunoDNA Washer, TBST or PBST buffer.
2. Perform PIER, proteolytic Digestion with Mohs ImmunoDigestor for 1 min. at room temperature.
3. Rinse with ImmunoDNA Washer, TBST or PBST buffer.

IHC Detection PRocedure

1. After PIER, transfer slides to ImmunoDNA washer and let it stand for 1-2 minutes.
2. For manual staining, perform antibody incubation at ambient temperature. For automated staining methods, perform antibody incubation according to instrument manufacturer’s instructions.
3. Wash slides with ImmunoDNA washer or DI water.
4. Continue IHC detection protocol. Wash slides between each step with ImmunoDNA washer solution.

Abbreviated Mohs PolyDetector DAB HRP Brown or HRP Green Immunohistochemical Protocol

1. Incubate slides with Peroxidase Blocker for 30 seconds
2. Wash with Buffer (TBST or PBST)
3. Incubate with Primary Antibody for 4 min
4. Wash with Buffer (TBST or PBST)
5. Incubate with HRP Label for 3 min
6. Wash with Buffer (TBST or PBST)
7. Prepare
   - DAB Brown (1 drop of DAB Chromogen in 1ml of DAB Buffer; mix well)
   - or HRP Green (1 drop of HRP Green Chromogen in 1 ml of HRP Green Buffer; mix well)
8. Incubate with DAB or HRP Green for 2 min
9. Wash with Buffer (TBST or PBST)
10. Counterstain with Hematoxylin or Nuclear Fast Red for 30 seconds
11. Wash with Buffer (TBST or PBST)
12. Mount with AquaMounter or dehydrate the tissue with Fast ChromoProtector then mount with PermaMounter

Step	Mohs PolyDetector HRP Green or DAB Protocol
Peroxidase Blocker	0.5 min.
Primary Antibody	4 min.
1st Step Detection	3 min.
Substrate-Chromogen	1-2 min.
Counterstain / Coverslip	Varies

This protocol can also be used with FFPE tissues retrieved with Citrate or EDTA.