**Rat Follicle Stimulating Hormone (FSH) Assay Procedure**
1. **Preparation of Standard Curve:**
Place 10 standard wells on the microplate. Add 100 μL of the standard solution to the first and second wells. Then, add 50 μL of the 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. Next, add 50 μL of the standard diluent to each of the third and fourth wells, mix well, and discard 50 μL from both. Then, add 50 μL of the mixture from the third and fourth wells into the fifth and sixth wells, followed by adding 50 μL of the standard diluent to each. Mix again. Repeat this process by transferring 50 μL from the fifth and sixth wells into the seventh and eighth wells, then adding 50 μL of the diluent to each. Finally, transfer 50 μL from the seventh and eighth wells into the ninth and tenth wells, and add 50 μL of the diluent to each. After mixing, remove 50 μL from the ninth and tenth wells. Each well now contains 50 μL 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 (no sample or enzyme reagent added, but all other steps are the same). For the sample wells, add 40 μL of sample diluent, followed by 10 μL of the sample. This results in a 5-fold dilution. Carefully pipette the sample to the bottom of the well without touching the sides, and gently mix.
3. **Incubation:**
Seal the plate with a film and incubate at 37°C for 30 minutes.
4. **Washing Solution Preparation:**
Dilute the 20× washing buffer with distilled water to a 1× concentration and set aside.
5. **Washing Steps:**
Remove the sealing film, discard the liquid, and blot dry. Fill each well with the washing solution, let it stand for 30 seconds, then discard. Repeat this process five times. Gently pat the plate dry.
6. **Enzyme Reagent Addition:**
Add 50 μL of the enzyme-labeled reagent to each well except the blank wells.
7. **Second Incubation:**
Seal the plate and incubate at 37°C for another 30 minutes.
8. **Second Washing:**
Follow the same washing procedure as step 5.
9. **Color Development:**
Add 50 μL of color developer A, followed by 50 μL of color developer B. 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 that the measurement is performed within 15 minutes after adding the stop solution. Always zero the instrument using a blank well before starting the readings.
This detailed procedure ensures accurate quantification of FSH levels in rat serum or plasma samples. Make sure all steps are carried out carefully to maintain assay precision and reliability.
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