TITLE: U Michigan - Podocyte Counting And Density Analysis
AUTHORS: Jeff Hodgin, Jharna Saha

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span style = "font-weight:bold;">Summary: </span></div><div class = "text-block">This protocol is used to stain and count podocyte nuclei in mouse glomeruli for diabetic nephropathy study.</div></div>

[STEPS]
?. Protocol 1: Podocyte Nuclei Staining with Wilms’Tumor-1 (WT-1) Protein:1) Bake slides for 10 minutes at 60ºC if it is not baked previously 2) Wash 2x in xylene for 5 minutes 3) Wash 2x in 100% EtOH for 3 minutes 4) Wash 1x in 95% EtOH for 3 minutes 5) Wash 1x in 70% EtOH for 3 minutes 6) Rinse with dH²O 7) Place in PT Link contain 1x antigen retrieval reagent, pH 9, incubate at 97ºC for 20 minutes, and then wait until machine temperature reach to 70ºC. 8) Wash in TBST for 5 minutes 9) Use Pap Pen to encircle tissue 10) Block with Peroxidase 1 for 5 minutes 11) Wash 2x in 1x TBST for 2 minutes 12) Block with Background Sniper for 5 min 13) Wash 2x in TBST for 2 minutes 14) Dilute WT-1 antibody in Da Vinci Green diluent (1:200) 15) Incubate for 2 hours at room temperature in a humidified chamber. 16) Wash 2x in TBST for 2 minutes 17) Incubate with Mach 3 Rabbit Probe for 20 minutes 18) Wash 2x in TBST for 2 minutes 19) Incubate with Mach 3 Rabbit HRP Polymer for 20 minutes 20) Wash 2x in TBST for 3 minutes21) Develop with Betazoid DAB-CHROMOGENK for 5 minutes, place slide under the microscope to check staining intensity 22) Stop reaction in dH²O 23) Counterstain with Tacha’s Hematoxylin for 3 minutes 24) Rinse in dH²O to check staining intensity – reapply hematoxylin if not dark enough 25) Rinse in TBST to blue 26) Wash with dH²O 27) Wash in 70% EtOH for 2 minutes 28) Wash in 95% EtOH for 2 minutes 29) Wash 2x in 100% EtOH for 2 minutes 30) Wash in xylene for 8 minutes 31) Mount slides with mounting medium -1 drop 32) Insert cover glass carefully, avoid bubble
?. Protocol 2: Podocyte Counting and Density Analysis:Pre-Operating Instructions: Camera and Microscope should be calibrated and values loaded into MetaMorph® Program.1) Photograph 50 consecutive glomerular cross-sections moving systematically from outer cortex to inner cortex and back so as to provide an equal sample from all cortical regions. Use phase contrast which gives podocyte nuclei golden color that makes them easier to count. 2) From these photomicrographs (which contain 1-3 glomeruli per photograph in about 30 photographs), measure the glomerular area by using Metamorph Image Analysis Software (version 6.1 ) and count the podocytes in 50 sequential glomerular cross-sections at two thicknesses (3 and 9 microns). 3) Measure the glomerular area by using Metamorph software (camera and microscope should be calibrated and values should be loaded into Metamorph program before outlining the glomerular tuft area). Click on the desktop icon (metamorph software icon) to open the metamorph program, and then open the images. From the menu bar, select Measure, then select Calibrate Distance. A calibration window will appear. Highlight the 40x calibration and then click on Apply. Use Polygon tool from menu bar for outlining the glomerular tuft area. From the menu bar, select Measure and then Region Measurement to the measurement of the glomerular tuft area. 4) Use a systematic method (large size cut-off) which helps to count WT-1 positive podocyte nuclei but eliminates false counting of granules. From the menu bar, choose Measure, then Manually Count Objects. Select the number 6. Use this size restriction method to check if the number 6 from the Metamorph Image Analysis System fits within the nuclear profile. If so, count it. If the number 6 was larger than the WT-1 positive area, do not count it. Data Analysis:1) Count the podocyte numbers (P) and measure the glomerular tuft area (GA) from 100 consecutive cross sections per animal (50 from thick and 50 from thin section) then calculate the average for each set of 50. 2) Divide the average podocyte number (P) by the average glomerular area (GA) to get podocyte number per glomerular area (P/GA) for both 3 and 9 micron sections. 3) The difference between the P/GA of the thick and the P/GA of the thin sections yielded the P/GA Δ which is directly related to the actual difference in section thickness (6.3um). 4) Calculate the average glomerular volume per podocyte (GV/P) by dividing the actual section thickness of 6.3 by the P/GAΔ. 5) Determine the glomerular volume by using the Weibel formula First, calculate the glomerular radius of both thick and thin sections by assuming circular cross sections using formula radius R = (GA/π)1/2and then calculate the average radius (Rav) that yielded the average maximum radiusRmax = 4Rav/π. Then calculate average glomerular volume as GV = 4/3π(Rmax) 3Then divide the average glomerular volume by the average volume per podocyte (GV/P) to get the podocyte number per glomerulus:P = GV/(GV/P)