Reagents

• Filter (Gelman GN6 13 mm) or (Millipore RAW 013 )
• Filter holder sweeny 13 mm
• Hank's solution
• Hématoxiline acide free
• Orange G ( Ortho )
• Ea 65 ou Ea 50 ( Ortho )
• EtOH 95%, 1-propanol, toluène

Acetate filter procedure

1. Centrifuge 10 ml of urine
2. Decant a maximum of supernatant
3. Resuspend the pellet to 1 ml with Hank's solution. Verify the cellularity. Dilute if necessary
4. Bring to volume to its initial value using Hank. Mix
5. Place the acetate filter in ethanol 95% for 10-15 sec, mount on the sweeny filter holder.
6. Rinse the filter by injecting 2 ml of Hank
7. Inject with a seringe, 1ml or less of the sediment suspension, depending on the cellularity.
8. Rinse the filter by injecting 2 to 5 ml of Hank
9. Un-mount the holder and retrieve the filter.
10. Fix immediately with EtOH 95%. Never let the filter dry out.
11. Stain and mount.

PAP stain for acetate filter

From: Barwick H. McGill University ( personnal communication)

1. EtOH 95%............................10 min.
2. H2O ....................................10 min.
3. Hematoxiline** .....................2 min.
4. H2O tap ..............................10 min.
5. HCl 0.05% ..........................7 dips (destain as needed )
6. H2O tap 37°C .....................6 min.
7. EtOH 95% ...........................2 min.
8. Orange G** ........................2 min.
9. EtOH 95% ..........................2 min.
10. EtOH 95% ..........................2 min.
11. EtOH 95% ..........................2 min.
12. EA 65**...............................2 min.
13. EtOH 95%............................2 min.
14. EtOH 95%............................2 min.
15. EtOH 95%............................2 min.
16. 1-propanol 100%..................2 min.
17. 1-propanol 100%..................2 min.
18. 1-propanol /Toluène ( 1:1).....2 min.
19. Toluène..................................6 min.

Mount

1. Slide
2. 1 drop of Eukitt
3. Put the filter on the drop
4. 2 to 4 drops of Eukitt on the filter
5. Add the coverslip

Put on a heating plate adjusted at 50C, and let the resin disperse.

Results

This procedure is used for collection purpose.

 

 

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