EnVision FLEX+, Mouse, High pH (Link) Visualization System

Intended Use

For in vitro diagnostic use. The EnVision FLEX+ visualization system is intended for automated laboratory use in qualitative or semiquantitative immunohistochemistry with Autostainer Link instruments. It detects primary mouse and rabbit antibodies in formalin-fixed, paraffin-embedded (FFPE) tissue sections. Antibody/antigen complexes are visualized using EnVision FLEX DAB+ Chromogen. Using EnVision FLEX+ Mouse (LINKER) (Code K8021) or EnVision FLEX+ Rabbit (LINKER) (Code K8009) can amplify signal for mouse or rabbit primary antibodies, respectively.

Summary and Explanation

The EnVision FLEX+ system is designed for flexible visualization of antibody/antigen complexes, providing optimal staining on Autostainer Link instruments when following the recommended protocol. EnVision FLEX+ Mouse (Code K8002) typically increases signal amplification 4-5 fold, while EnVision FLEX+ Rabbit (Code K8009, optional) increases it 2-3 fold. Before staining, FFPE tissue sections require deparaffinization, hydration, and heat-induced epitope retrieval (HIER) using the primary antibody's specified method. Code K8002 includes EnVision FLEX Target Retrieval Solution, High pH (50x). Alternatives include EnVision FLEX Target Retrieval Solution, High pH (50x) (Code K8004) or Low pH (50x) (Code K8005). Some antibodies may require enzymatic pre-treatment instead of HIER. Endogenous peroxidase blocking is performed with the included EnVision FLEX Peroxidase-Blocking Reagent (SM801). Effective washing and stabilizing proteins make extra blocking steps unnecessary. EnVision FLEX Wash Buffer (20x) (DM831 or optional K8007) is recommended. Primary antibodies are not included; Agilent's FLEX Ready-to-Use or concentrated primary antibodies are recommended. EnVision FLEX Antibody Diluent (Code K8006) is for diluting concentrated primary antibodies. EnVision FLEX+ Mouse (LINKER) (DM824 or optional K8021) can substitute for Rabbit (LINKER) (Code K8009) when using rabbit primary antibodies. The EnVision FLEX/HRP visualization reagent (SM802) uses a dextran backbone coupled with peroxidase and secondary antibodies. The substrate system comprises EnVision FLEX DAB+ Chromogen (DM827) and EnVision FLEX Substrate Buffer (SM803) with hydrogen peroxide. The substrate system produces a crisp brown end product at the target antigen site. EnVision FLEX Hematoxylin (Code K8008) is recommended for counterstaining, providing clear blue nuclear staining.

Reagents

Materials Provided

Optional Reagents

Other Options

Precautions

• For in vitro diagnostic use.
• For professional users only.
• Prescription use only (Rx Only).
• Products contain material of animal origin; trace human material cannot be excluded due to manufacturing. Handle with appropriate care according to local requirements.
• Do not expose Peroxidase-Blocking Reagent, /HRP, Substrate Buffer, DAB+ Chromogen, or Substrate Working Solution to strong light.
• Safety data sheets are available on www.agilent.com or upon request.
• Substrate Buffer contains an endocrine disruptor. Dispose of unused solution and waste according to all local, regional, national, and international regulations.
• Dispose of other vials' unused solution according to all local, regional, national, and international regulations.
• EnVision FLEX Target Retrieval Solution, High pH (50x) is labeled with warnings for serious eye irritation. Wear eye/face protection. In case of eye contact, rinse cautiously with water for several minutes, removing contact lenses if present and easy to do. Continue rinsing. Seek medical advice if irritation persists. ⚠ Warning
• EnVision FLEX Wash Buffer (20x) is labeled with warnings for serious eye irritation. Wear eye/face protection. In case of eye contact, rinse cautiously with water for several minutes, removing contact lenses if present and easy to do. Continue rinsing. Seek medical advice if irritation persists. ⚠ Warning
• EnVision FLEX Peroxidase-Blocking Reagent and EnVision FLEX /HRP are labeled: Safety data sheet available on request.
• EnVision FLEX Substrate Buffer is labeled: 🗻 Danger Contains imidazole. May damage the unborn child. Obtain special instructions before use. Wear protective gloves, clothing, eye/face protection. Seek medical advice if exposed or concerned. Dispose of contents/containers according to regulations.
• EnVision FLEX DAB+ Chromogen is labeled: 🗻 Danger Contains 3,3'-diaminobenzidine tetrahydrochloride. Suspected of causing genetic defects. May cause cancer. Obtain special instructions before use. Wear protective gloves, clothing, eye/face protection. Seek medical advice if exposed or concerned. Dispose of contents/containers according to regulations.
• Products contain sodium azide (NaN3), which can react with lead and copper plumbing to form explosive metal azides. Flush with large volumes of water upon disposal.
• Wear appropriate Personal Protective Equipment (PPE) to avoid eye and skin contact.
• Contact Agilent Pathology Support (www.agilent.com) for unusual staining. Report serious incidents to the manufacturer and competent authority.
• Persons under 18 years of age are not permitted to work with this product. Users must be trained on proper procedures, hazards, and safety instructions. Refer to SDS for more information.

Storage

Store EnVision FLEX and EnVision FLEX+ reagents at 2–8 °C. Store Peroxidase-Blocking Reagent, /HRP, and Substrate Buffer in the dark at 2–8 °C. Do not use after expiration date. Verify storage conditions if different from specified. Keep caps closed when not in use on Autostainer Link. Store prepared Substrate Working Solution in the dark at 2–8 °C and use within 5 days.

Reagent Preparation

No obvious signs of instability. Run positive and negative controls simultaneously with patient specimens. Contact Agilent Pathology Support for unexpected staining not explained by lab procedures.

A.1 EnVision FLEX Target Retrieval Solution, High pH (50x) (DM828, Code K8004)

Dilute 1:50 with distilled or de-ionized water. For PT Link pre-treatment, transfer vial contents to PT Link tank and fill to marked line with water. Unused diluted solution: store at 2–8 °C for 1 month; discard if cloudy. For 3-in-1 procedure in PT Link, diluted solution can be used 3 times within 5 days if stored at room temperature (20–25 °C).

A.2 EnVision FLEX Target Retrieval Solution, Low pH (50x) (DM829, Code K8005)

Dilute 1:50 with distilled or de-ionized water. For PT Link pre-treatment, transfer vial contents to PT Link tank and fill to marked line with water. Unused diluted solution: store at 2–8 °C for 1 month; discard if cloudy. For 3-in-1 procedure in PT Link, diluted solution can be used 3 times within 5 days if stored at room temperature.

A.3 EnVision FLEX Wash Buffer (20x) (DM831, Code K8007)

Dilute 1:20 with distilled or de-ionized water. Add concentrated buffer to pre-measured water to minimize foaming. Stir gently until homogenous. Unused diluted solution: store at 2–8 °C for 1 month; discard if cloudy. For PT Link Rinse Station wash step after 3-in-1 procedure, diluted solution can be used 3 times within 5 days if stored at room temperature.

A.4 EnVision FLEX Substrate Working Solution

Mix 1 drop EnVision FLEX DAB+ Chromogen (DM827) with 1 mL EnVision FLEX Substrate Buffer (SM803). Use within 5 days (store in dark at 2–8 °C).

Specimen Collection and Preparation

Specimens: formalin-fixed, paraffin-embedded tissue sections. Fixation time depends on fixative and tissue type/thickness (e.g., 3–4 mm blocks in neutral-buffered formalin for 18–24 hours). Optimal section thickness: ~4 µm. After cutting, mount tissues on FLEX IHC Microscope Slides (K8020) or SuperFrost Plus slides. Place in a calibrated oven at 58 ± 2 °C for 1 hour. FLEX IHC Microscope Slides (K8020) are recommended for better adherence. Mount sections flat and wrinkle-free; wrinkles negatively impact staining. NOTE: Microscope slide width must be suitable for Autostainer Link. Refer to Agilent instrument User Guide for compatible slides.

Procedure

Read the Agilent instrument User Guide and PT Link User Guide before running protocols. Load slide holder fully for reproducible epitope retrieval and uniform heating.

A. Pre-treatment procedure

Recommended 3-in-1 specimen preparation using PT Link:

Deparaffinization, rehydration, and HIER on FFPE tissue sections:

1. Prepare working solution: dilute EnVision FLEX Target Retrieval Solution (50x) 1:50 in distilled/de-ionized water.
2. Fill PT Link tanks with 1.5 L of working solution.
3. Set PT Link to pre-heat solution to 65 °C.
4. Immerse mounted FFPE sections in pre-heated EnVision FLEX Target Retrieval Solution (working solution) in PT Link tanks. Incubate for 20–40 minutes at 97 °C (optimal time determined by user).
5. Cool sections in PT Link to 65 °C.
6. Remove slide racks from PT Link and immediately dip slides into a jar/tank (e.g., PT Link Rinse Station, Code PT109) with diluted, room temperature EnVision FLEX Wash Buffer (20x).
7. Leave slides in diluted Wash Buffer for 1–5 minutes.
8. Place slides on Autostainer Link and proceed with staining. Do not let sections dry out.
9. After staining, perform dehydration, clearing, and permanent mounting.
10. NOTE: Diluted Target Retrieval Solution and Wash Buffer can be used 3 times within 5 days if stored at room temperature.

HIER procedure using PT Link:

After deparaffinization and hydration to buffer (water), perform HIER:

1. Prepare working solution: dilute EnVision FLEX Target Retrieval Solution (50x) 1:50 in distilled/de-ionized water.
2. Fill PT Link tanks with 1.5 L of working solution.
3. Set PT Link to pre-heat solution to 65 °C.
4. Manually deparaffinize and rehydrate sections on slides. Coated or silanized slides recommended for better adhesion.
5. Immerse room temperature sections in pre-heated EnVision FLEX Target Retrieval Solution (working solution) and incubate for 20–40 minutes at 97 °C (optimal time determined by user).
6. Cool sections in PT Link to 65 °C.
7. Remove slide racks from PT Link and immediately dip slides into a jar/tank (e.g., PT Link Rinse Station, Code PT109) with diluted, room temperature EnVision FLEX Wash Buffer (20x).
8. Leave slides in diluted Wash Buffer for 1–5 minutes.
9. Place slides on Autostainer Link and proceed with staining. Do not let sections dry out.
10. Mount slides using aqueous or permanent mounting medium after staining.
11. NOTE: Diluted Target Retrieval Solution and Wash Buffer can be used 3 times within 5 days if stored at room temperature.

HIER procedure using Coplin jars:

After deparaffinization and hydration, perform HIER:

1. Place staining jars with diluted EnVision FLEX Target Retrieval Solution (50x) in a water bath.
2. Heat water bath and jars to 95–99 °C. Cover jars to stabilize temperature and prevent evaporation.
3. Immerse room temperature sections in preheated EnVision FLEX Target Retrieval Solution (working solution) in staining jars.
4. Re-equilibrate water bath and solution temperature to 95–99 °C. Incubate for 20 (±1) minutes at 95–99 °C.
5. Remove jar with slides from water bath. Allow slides to cool in EnVision FLEX Target Retrieval Solution for 20 (±1) minutes at room temperature.
6. Decant EnVision FLEX Target Retrieval Solution and rinse sections with diluted, room temperature EnVision FLEX Wash Buffer (20x) for 1–5 minutes.
7. Place rack with rinsed sections on Autostainer Link and start programmed staining run.
8. NOTE: For Coplin jar HIER, diluted Target Retrieval Solution and Wash Buffer are for single use only.

Some epitopes may not tolerate HIER and require enzymatic pre-treatment. Refer to the primary antibody's Instructions for Use. Do not allow tissue sections to dry out during treatment or staining. FLEX IHC Microscope Slides (K8020) recommended for better adherence.

B. Staining procedure

FLEX Ready-to-Use primary antibodies can be used with EnVision FLEX+ on FFPE tissue sections. Dilution guidelines for concentrated primary antibodies are in Agilent's Instructions for Use. Staining steps and incubation times are pre-programmed in Autostainer Link software for protocols like FLEX, FLEX+ Mouse (LINKER), and FLEX+ Rabbit (LINKER). Recommended reagent volume: 1 x 200 µL or 2 x 150 µL per slide. Contact Agilent Pathology Support if protocols are unavailable. Optimal incubation times for primary antibody and EnVision FLEX/HRP depend on the primary antibody; refer to its Instructions for Use. User must verify the protocol. After staining, mount specimens. Dehydration, clearing, and permanent mounting recommended for 3-in-1 procedure. For HIER procedure, aqueous or permanent mounting medium can be used. Glycergel (C0563) or Faramount Aqueous Mounting Medium (S3025) recommended for aqueous mounting. Do not let sections dry before mounting.

Quality Control

Each run should include a positive control specimen to verify reagent performance. If positive control fails, test specimens are invalid. Use a negative control reagent to identify nonspecific staining. If nonspecific staining cannot be differentiated from specific staining, test specimens are invalid. Positive/negative control tissue and reagent should be tested simultaneously with patient specimens on the same slide for validity.

Staining Evaluation

The DAB-containing substrate working solution produces a brown color at the target antigen site. Brown color should be present on the positive control at the expected location. Diffuse brown staining on slides treated with negative control reagent indicates nonspecific staining. Nuclei are stained blue by hematoxylin counterstain.

Cross-reactivity of Secondary Antibodies

Goat anti-mouse antibody in EnVision FLEX/HRP reacts with mouse IgG subclasses, IgA, and IgM. Cross-reactions with human, cow, rabbit immunoglobulins are <1%. Fetal calf serum <0.1%. Other cross-reactions: Guinea pig ~8%, swine ~4%, rat ~30%. Human serum cross-reaction <0.1%. Goat anti-rabbit antibody reacts with rabbit immunoglobulins of all classes. Cross-reactions with human, rat, mouse immunoglobulins are <1%. Bovine immunoglobulins and fetal calf serum <0.1%. Other cross-reactions: Guinea pig ~10%, swine ~1%. Human serum cross-reaction <0.1%.

Limitations

1. For automated use on Autostainer Link instruments only. Not validated for manual staining.
2. For formalin-fixed, paraffin-embedded tissue sections only. Not validated for frozen sections or cell smears.
3. Procedure modifications may affect the number of tests provided.

Explanation of Symbols

Symbols used in this document include:

• [REF]: Catalogue number
• [IVD]: In vitro diagnostic medical device
• [⚠]: Consult instructions for use
• [🗻]: Caution/Danger
• [🌡]: Store in the dark
• [📅]: Temperature limitation
• [📈]: Batch code
• [🕓]: Use by
• [UDI]: Unique device identification
• [☾]: Contains biological material of animal origin
• [💾]: Manufacturer
• [💾]: Authorized representative in the European Community
• [🔬]: GHS pictogram (consult precautions section)