TintoFast p40 (ZR8), RMab

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 for 2 minutes at room temperature.

Pretreatment of Mohs Frozen Tissues

1. Preheat the TintoDetector Incubator to 110°C.
2. Place TintoDetector Cap Gap slides (BSB 7006) face to face and insert them into the TintoDetector Slide Holder (BSB 7003).
3. Submerge slides in ImmunoDNA Retriever with EDTA to draw up enough solution by capillary action to cover the tissues.
4. Heat the slides in a preheated TintoDetector Incubator for 3 minutes.
5. Transfer slides to room temperature and cool off for 1 min.

Mohs IHC Detection

1. After HIER, 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 Plus DAB HRP Brown or HRP Green Immunohistochemical Protocol

1. Incubate with Primary Antibody for 5 min
2. Wash with Buffer (TBST or PBST)
3. Incubate with M/R link for 4 min
4. Wash with Buffer (TBST or PBST)
5. HRP Label for 4 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 1-2 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 Plus HRP Green or DAB 20 Minute Protocol
HIER	3 min.
Primary Antibody	5 min.
1st Step Detection	4 min.
2nd Step Detection	4 min.
Substrate-Chromogen	1-2 min.
Counterstain / Coverslip	Varies

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