The Rat VEGF Immunohistochemistry Kit is designed for the detection of Vascular Endothelial Growth Factor (VEGF) in cells and tissues using a HRP-Streptavidin Conjugate (HRP-SA) system. This method offers high sensitivity, specificity, and clear background, ensuring accurate localization of VEGF antigens. The process involves the use of a primary antibody that binds specifically to VEGF, followed by a biotinylated secondary antibody that recognizes the primary antibody. Finally, the HRP-SA complex is added, forming a stable antigen-antibody-biotin-HRP-SA complex, which can be visualized under a microscope after DAB staining.
**Kit Components:**
- **Reagent A (Permeate):** 0.1% Triton-X 100 in 10 mL (optional)
- **Reagent B (Blocking Buffer):** 20 mL for blocking non-specific sites
- **Reagent C (Primary Antibody):** Ready-to-use VEGF Antibody (2.5 mL)
- **Reagent D (Secondary Antibody):** Biotinylated Goat Anti-Rabbit IgG (1.5 mg/mL), diluted 1:300–1:500 (50 μL + 20 mL diluent)
- **Reagent E (HRP-SA Complex):** 1 μM concentration, diluted 1:50–1:200 (100 μL)
- **Reagent F (DAB Substrate):** 5 mL for color development
**User-Supplied Reagents:**
1. **TBS (10 mM, pH 7.2–7.4):** 1.21 g Tris, 7.6 g NaCl in 800 mL water; adjust pH with HCl to 7.2–7.4, then bring to 1 L.
2. **TBS-T (0.05% Tween 20):** TBS with 0.05% Tween 20 added.
3. **Antigen Retrieval Solution:**
- Citrate buffer (pH 6.0): 0.38 g citric acid, 2.45 g trisodium citrate in 900 mL water, adjust pH to 6.0 with HCl, then bring to 1 L.
- EDTA buffer (pH 8.0): 186.1 g EDTA·2H2O, 2.45 g trisodium citrate in 700 mL water, adjust pH with 10 mM NaOH to 8.0, then bring to 1 L.
4. **Mounting Medium:** 10 mL glycerin-based mounting medium
5. **Tween 20:** 5 mL for washing steps
**Immunostaining Procedure (Paraffin-Embedded Tissue):**
1. **Section Preparation:** Cut 3–4 μm thick sections and bake at 60°C for 1 hour.
2. **Deparaffinization:** Soak in xylene three times (10 min each).
3. **Hydration:** Gradually rehydrate through descending ethanol concentrations (anhydrous ethanol 5 min, 95% ethanol 2 × 2 min, 85%, 75% ethanol 2 min each).
4. **Antigen Retrieval:** Use either microwave, pressure cooker, or enzymatic digestion depending on the target antigen. Cool naturally, rinse with tap water and ddH2O, then wash with TBS.
5. **Blocking:** Incubate in Reagent B for 30 min at 37°C.
6. **Primary Antibody Incubation:** Add Reagent C and incubate at 37°C for 2 hours or overnight at 4°C.
7. **Washing:** Rinse with TBS-T three times (5 min each).
8. **Secondary Antibody Incubation:** Dilute Reagent D in antibody diluent and incubate for 30 min at 37°C.
9. **Washing:** Rinse with TBS-T three times (5 min each).
10. **Blocking with Tween 20:** Incubate with Tween 20 for 20 min at 37°C.
11. **HRP-SA Incubation:** Dilute Reagent E (1:50–1:200) in antibody diluent and incubate for 30 min at 37°C.
12. **Washing:** Rinse with TBS-T three times and TBS twice (5 min each).
13. **Color Development:** Apply DAB solution (Reagent F) until desired staining is achieved.
14. **Counterstaining:** Wash with tap water, stain nuclei with hematoxylin, dehydrate, and clear.
15. **Mounting:** Seal with glycerin mounting medium once dry.
16. **Imaging:** Observe under a microscope.
**Notes:**
- Always cool antigen retrieval buffer naturally before washing.
- Ensure sufficient volume of buffer to cover all sections; do not reuse citrate buffer.
- If the reagent is concentrated, centrifuge briefly before use to remove any residue.
- Wash thoroughly with TBS before sealing to avoid residual Tween 20 interference.
- For nuclear counterstaining, either stain before sealing or use a mounting medium containing a nuclear dye.
**Antigen Retrieval Methods:**
1. **Enzymatic Digestion:** After dewaxing and hydration, incubate with pepsin or trypsin at 37°C for 20–30 minutes.
2. **Microwave Method:** Heat antigen retrieval solution in a microwave, place tissue sections in boiling buffer, microwave for 10–15 minutes, cool, and wash.
3. **Pressure Cooker Method:** Boil retrieval solution, place tissue in a heat-resistant rack, apply pressure for 1.5–2.5 minutes, then cool naturally.
4. **Combined Microwave & Pressure Method:** Use a combination of microwave and pressure for more challenging antigens, typically 4–8 minutes under pressure.
This detailed protocol ensures reliable and reproducible results when detecting VEGF in paraffin-embedded tissues.
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