Aug 07, 2019
Version 2
U Michigan - Podocyte Counting And Density Analysis V.2
- Jeff Hodgin1,
- Jharna Saha1
- 1University of Michigan - Ann Arbor
- Mouse Metabolic Phenotyping CentersTech. support email: info@mmpc.org
Protocol Citation: Jeff Hodgin, Jharna Saha 2019. U Michigan - Podocyte Counting And Density Analysis. protocols.io https://dx.doi.org/10.17504/protocols.io.565g9g6
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: August 05, 2019
Last Modified: August 07, 2019
Protocol Integer ID: 26557
Keywords: Podocyte Counting And Density Analysis, diabetic nephropathy study
Abstract
Summary:
This protocol is used to stain and count podocyte nuclei in mouse glomeruli for diabetic nephropathy study.
Materials
MATERIALS
MACH3 Rabbit HRP-Polymer Detection Kit Biocare MedicalCatalog #M3R531L
Da Vinchi Green DiluentBiocare MedicalCatalog #PD900L
Peroxidase 1 Biocare MedicalCatalog #PX968H
Betazoid DAB-CHROMOGENK KitBiocare MedicalCatalog #BDB2004L
Tachas Auto HematoxylinBiocare MedicalCatalog #NM-HEM
Background SnipperBiocare MedicalCatalog #BS966
XyleneFisher ScientificCatalog #X3P-1GAL
Ethanol (EtOH) 200 ProofDecon LaboratoriesCatalog #2701
ImmEdge hydrophobic barrier pap penVector LaboratoriesCatalog #H-4000
Humidified chamber Catalog #Customized by lab. personnel
Mounting MediumThermo ScientificCatalog #8312-4
Cover GlassFisher ScientificCatalog #12-542-B
PT Link ModuleThermo ScientificCatalog #A80400012
Universal Imaging Metamorph Imaging System Molecular Devices
Scientific grade digital color CCD camera RT SLIDER DIAGNOSTIC
Perfusion-fixed paraffin embedded sections
Microscope and LenseLeica MicrosystemsCatalog #Leica DM IRB
Reagent Preparation:
Reagent 1: 20x TBST Wash Buffer (working concentration 1x)
Reagents and Materials: Tris-base, Sodium chloride, Tween-20, HCl, Distilled water
Procedure:
● Tris-base 48.4g
● Sodium chloride 160g
● Tween-20 10 ml
● Mix to dissolve
● Adjust pH 7.4 with concentrated hydrochloric acid (HCl) before add Tween-20
● Distilled water up to 1L
● Store at room temperature
NOTE:
Biocare Medical, RRID: SCR-013549
Thermo Fisher Scientific/ Fisher Scientific, RRID:SCR_008452
Vector Laboratories, RRID:SCR_000821
Vector Laboratories Cat# H-4000, RRID:AB_2336517
Leica Microsystems, RRID:SCR_008960
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 minutes
21) 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)1/2
and then calculate the average radius (Rav) that yielded the average maximum radius
Rmax = 4Rav/π.
Then calculate average glomerular volume as
GV = 4/3π(Rmax) 3
Then divide the average glomerular volume by the average volume per podocyte (GV/P) to get the podocyte number per glomerulus:
P = GV/(GV/P)