**Rat Follicle Stimulating Hormone (FSH) Assay Procedure**
1. **Preparation of Standard Curve:**
Place 10 standard wells on the microtiter plate. Add 100 µL of standard solution to the first and second wells. Then, add 50 µL of standard diluent to the second well and mix thoroughly. Transfer 100 µL from the first and second wells into the third and fourth wells, respectively. Add 50 µL of standard diluent to each of the third and fourth wells, mix again, and discard 50 µL from both. Next, transfer 50 µL from the third and fourth wells into the fifth and sixth wells, then add 50 µL of standard diluent to each and mix. Repeat this serial dilution process until the tenth well. After completing the dilutions, each well contains 50 µL of solution with concentrations of 18 U/L, 12 U/L, 6 U/L, 3 U/L, and 1.5 U/L, respectively.
2. **Sample Addition:**
Set up blank control wells by not adding any sample or enzyme reagent, but following all other steps. For the test samples, add 40 µL of sample diluent to each sample well, followed by 10 µL of the sample. This results in a 5-fold dilution. Carefully pipette the sample into the bottom of each well without touching the walls, and gently mix by tapping the plate.
3. **Incubation:**
Seal the plate with an adhesive film and incubate at 37°C for 30 minutes.
4. **Washing Solution Preparation:**
Dilute the 20× washing buffer with distilled water to make a 1× working solution and set aside.
5. **Washing Steps:**
Remove the sealing film, discard the liquid, and blot dry. Fill each well with the 1× washing solution, let it stand for 30 seconds, then discard. Repeat this process five times. Gently pat the plate dry before proceeding.
6. **Enzyme Conjugate Addition:**
Add 50 µL of enzyme-labeled reagent to each well except for the blank wells.
7. **Second Incubation:**
Seal the plate again and incubate at 37°C for another 30 minutes.
8. **Second Washing:**
Follow the same washing procedure as step 5: five washes with 1× washing solution.
9. **Color Development:**
Add 50 µL of color reagent A, followed by 50 µL of color reagent B to each well. Mix gently and incubate at 37°C for 15 minutes in the dark.
10. **Stop Reaction:**
Add 50 µL of stop solution to each well to terminate the reaction. The color will change from blue to yellow.
11. **Absorbance Measurement:**
Measure the absorbance at 450 nm using a microplate reader. Ensure the measurement is performed within 15 minutes after adding the stop solution. Always zero the instrument using the blank well before starting the readings.
This detailed procedure ensures accurate quantification of FSH levels in rat serum or plasma samples. Proper execution of each step is critical for reliable results.
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