**Rat VEGF Immunohistochemistry Kit Instructions**
This Rat VEGF Immunohistochemistry Kit is designed for the detection of Vascular Endothelial Growth Factor (VEGF) in cells and tissues using a horseradish peroxidase (HRP)-conjugated streptavidin (HRP-SA) system. The kit offers high sensitivity, strong specificity, precise localization, and a clean background, making it ideal for accurate immunostaining analysis.
The process involves the use of a primary antibody that specifically binds to the VEGF antigen, followed by a biotinylated secondary antibody that recognizes the primary antibody. Finally, HRP-SA is added to form a stable complex, which can be visualized under a microscope after the DAB staining reaction.
**Kit Components:**
- **Reagent A (Permeate):** 0.1% Triton-X 100, 10 mL (optional)
- **Reagent B (Blocking Buffer):** 20 mL
- **Reagent C (Primary Antibody):** Ready-to-use VEGF Anti, 2.5 mL
- **Reagent D (Biotinylated Secondary Antibody):** Goat anti-rabbit IgG (1.5 mg/mL), diluted 1:300–1:500, 50 μL + 20 mL antibody diluent
- **Reagent E (HRP-SA Complex):** 1 μM, diluted 1:50–1:200, 100 μL
- **Reagent F (DAB Substrate):** 5 mL
**User-Supplied Reagents:**
1. **TBS (pH 7.2–7.4):** 10 mM Tris-HCl, 150 mM NaCl, 0.05% Tween 20 (TBS-T)
2. **Antigen Retrieval Solution:**
- Citrate buffer (pH 6.0): 0.01 M citric acid, 0.05 M trisodium citrate
- EDTA buffer (pH 8.0): 0.5 M EDTA·2H₂O, 0.05 M trisodium citrate
3. **Mounting Medium:** Glycerin-based, 10 mL
4. **Tween 20:** 5 mL
**Immunostaining Procedure for 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 for 10 minutes each.
3. **Hydration:** Gradually rehydrate through descending alcohol concentrations (anhydrous ethanol 5 min, 95% ethanol ×2, 85%, 75%), then rinse with tap water and ddH₂O.
4. **Antigen Retrieval:** Follow the recommended method for the specific antibody. Common methods include:
- **Microwave:** Heat antigen retrieval solution to boiling, place tissue in the solution, microwave for 10–15 minutes.
- **Pressure Cooker:** Boil retrieval solution, place tissue in the cooker, heat until steam releases, then cool naturally.
- **Enzymatic Digestion:** Use pepsin or trypsin at 37°C for 20–30 minutes.
5. **Blocking:** Incubate with Reagent B for 30 minutes at 37°C.
6. **Primary Antibody Incubation:** Apply Reagent C and incubate at 37°C for 2 hours or overnight at 4°C.
7. **Washing:** Rinse with TBS-T three times for 5 minutes each.
8. **Secondary Antibody Incubation:** Dilute Reagent D in antibody diluent and incubate for 30 minutes at 37°C.
9. **Washing:** Rinse with TBS-T three times and TBS twice.
10. **Blocking with Tween 20:** Incubate with Tween 20 for 20 minutes at 37°C.
11. **HRP-SA Incubation:** Dilute Reagent E (1:50–200) and incubate for 30 minutes.
12. **Washing:** Rinse with TBS-T three times and TBS twice.
13. **DAB Staining:** Apply DAB solution and monitor development.
14. **Counterstaining:** Wash with tap water, stain nuclei (e.g., hematoxylin), dehydrate, and clear.
15. **Mounting:** Seal with glycerin mounting medium.
16. **Observation:** Examine under a microscope.
**Notes:**
- Always allow antigen retrieval solutions to cool naturally before proceeding.
- Ensure sufficient buffer volume to cover all sections; do not reuse buffers.
- If the reagent is a concentrate, centrifuge briefly before use to avoid loss.
- Wash thoroughly with TBS before sealing to remove any residual Tween 20.
- For nuclear counterstaining, either pre-stain or use a mounting medium containing a nuclear dye.
**Common Antigen Retrieval Methods:**
1. **Enzymatic Method:** After dewaxing and hydration, incubate in pepsin or trypsin at 37°C for 20–30 minutes.
2. **Microwave Method:** Place slices in antigen retrieval solution and microwave for 10–15 minutes.
3. **Pressure Cooker Method:** Boil retrieval solution, add tissue, and heat until pressure builds up.
4. **Water-Proof Pressure Method:** Combine microwave and pressure techniques for more challenging antigens.
This kit provides a reliable and efficient way to detect VEGF in paraffin-embedded tissues, suitable for both research and diagnostic applications.
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