Our protocol observes that the use of kidney extracts from cows and pigs are an excellent and economical resource for the study of renal physiology, particularly examining the correlation between vascular endothelial growth factor and other analytes. It has always been a challenge to correlate VEGF, an essential growth factor in organ angiogenesis, with other proteins. This technique allows us to examine the association between VEGF and analytes such as hormones.
This will certainly advance our base of knowledge regarding this important growth factor. This method may be useful for correlating other analytes in kidney cortical extracts besides the ones mentioned in this video. Visual demonstration will help others to obtain reproducible results without any sampling errors.
The use of fresh tissue is essential to the success of this technique. It is important to obtain fresh whole kidneys immediately after slaughter from an animal. Always transfer the tissue to the laboratory on ice.
Use sterile scissors, forceps, a knife, and Petri dishes to dissect kidneys and excise the required tissue portion. Gently cut the kidney in half along the sagittal plane and cut a piece of tissue from the cortex region in the center of the kidney. Mince the tissue into small pieces with the knife and transfer it into a microfuge tube with one milliliter of ice cold 1X RIPA lysis extraction buffer.
Label the tube with specific sample details and place it on ice. Use a handheld homogenizer with a sterile probe to homogenize the tissue for one to two minutes until no chunks are visible, then immediately centrifuge the tube at 9, 600 times g for five minutes at four degrees Celsius. After centrifugation, prepare separate aliquots of the sample for ELISA and total protein analysis to avoid multiple freeze/thaw cycles.
Prior to starting the assay, bring all reagents and the assay plate to room temperature removing the excess wells and storing them at four degrees Celsius until further use. Dilute the 20X wash buffer to 300 milliliters with double distilled water and set up the blank wells without any solution. Add 50 microliters of standard or sample and another 50 microliters of horseradish peroxidase conjugate to each well, then immediately add 50 microliters of antibody solution to each well and seal the plate.
Mix the plate and incubate it at 37 degrees Celsius for one hour. After the incubation, wash each well with 200 microliters of wash buffer. Add 50 microliters of substrate A and substrate B to each well, mix the plate gently by tapping on the side, then seal and incubate it in the dark for 15 minutes at 37 degrees Celsius.
Add 50 microliters of stop solution to each well. Gently tap the plate and read the absorbance at 450 nanometers with a spectrophotometer. Prepare the reagents and wash buffer as previously described, then add 100 microliters of standard or sample to each well, seal the plate, mix well, and incubate it for two hours at 37 degrees Celsius.
Remove the liquid in each well and add 100 microliters of detection reagent A.Seal the plate and incubate it for another hour. Next, wash each well with 400 microliters of wash buffer, then add 90 microliters of substrate solution to each well, gently tap the plate, and incubate it for another hour at 37 degrees Celsius. After the last incubation, add 50 microliters of stop solution to each well, gently tap the plate, and read the absorbance at 450 nanometers with a spectrophotometer.
Estimate the total protein of the bovine and porcine kidney extracts with a standard BSA assay and use this estimate to normalize the LH and VEGF-A-ELISA results. The mean and median levels of LH and VEGF were calculated for both animals and sexes. After verifying normality of data, linear regression models were utilized to examine the relationship between LH and VEGF.
LH was found to be a strong and significant predictor of VEGF in both bovine and porcine kidneys. When attempting this procedure, it is important to remember to sample identical areas in each tissue. A similar procedure can be used to extract any type of tissue.
Remember to normalize analytes by total protein extract of whatever tissue you use.
