TITLE:
C4d Staining Protocol on formalin-fixed and paraffin
embedded tissue
(Envision+® PAP Method)
PURPOSE: To localize C4d staining in peritubular capillary basement membranes
and glomeruli for diagnosis of antibody mediated rejection
SPECIMEN: Formalin-fixed paraffin embedded section (2-4 microns)
MATERIAL: - BORG Decloaker (Biocare Medical® Cat.# BD1000MMM)
- Endogenous Peroxidase Blocking Reagent (DAKO® S2001)
- 10X Power Block Universal Blocking Reagent (BioGenex® Cat.#
HK085-5K)
- NGS Protein Blocking Reagent (BioGenex® Cat.# HK-112-9K)
- Primary Ab Rbhuman C4d (ALPCO Diagnostics® Cat.# 004-BIRC4d)
- Common Antibody Diluent CAD (BioGenex® Cat # HK-156-5K)
- Envision+ System labelled Polymer-HRP anti Rabbit (DAKO® Cat.#
K4000)
- Liquid DAB+ Substrate Chromogen (DAKO® K-33468)
- DAB Enhancer (DAKO® Cat.# S1961)
- Dako Wash Buffer 10X ( Dako Cat.# S3006 )
- Harris Modified Hematoxylin ( Fisher Scientific Cat.# SH26-500D)
- Xylene ( Fisher Scientific® Cat.# SN23100 )
- Ethyl Alcohol
EQUIPMENT:
- Pressure Cooker (Biocare Medical®)
- Humid slide chamber
METHOD:
1.
2.
3.
4.
5.
6.
7.
8.
9.
Deparaffinize and rehydrate tissue sections agitating slides 10X
each in the following: (2) changes Xylene 10min each, (2) 100%
ETOH, (1) 95%, (1) 75% ETOH, (1) 50% ETOH to water; (running
tap water until ETOH smell is gone)
Place slides in BORG Decloaker antigen retrieval solution and place
in Pressure Cooker containing 500mls of dH20, at 125ºC/30sec Set
Pt.1 and 90ºC/10sec Set Pt.2
Remove slides from Pressure Cooker unit and let cool ~ 20 min/RT
Rinse in dH20 then let stand in Dako Wash Buffer 5 min/RT
Incubate slides in 1X Power block 10min/RT
Rinse 3X with Dako Wash Buffer then let stand for 5 min/RT;
agitate slides 8X
Incubate in Protein Block 7min/37C
Do Not Rinse
Incubate slides in RbHuman C4d diluted 1/20 with CAD
30min/37C
10. Rinse 3X with Dako Wash Buffer then let stand for 5 min/RT;
agitate slides 8X
11. Incubate in Peroxidase Block 10min/RT
12. Rinse 3X with Dako Wash Buffer then let stand for 5 min/RT;
agitate slides 8X
13. Incubate in Envision+ System labelled Polymer-HRP anti Rabbit
30min/RT
14. Rinse 3X with Dako Wash Buffer then let stand for 5 min/RT;
agitate slides 8X
15. Incubate in DAB Substrate + Chromogen 1drop/ml of substrate
5min/37C
(quick check a slide every 2min to see the best signal to background
ratio for you)
16.Dip slides twice in Dako Wash Buffer to stop reaction; then 2X in
H20
17.Incubate in DAB Enhancer 2min/RT
18.Rinse in running tap water
19.Dip slides 12X in Harris Modified Hematoxylin
20.Rinse immediately in running water until water runs clear
21.Dip slides 12X in 0.2% Ammonium Hydroxide
22.Rinse in running water
23.Dehydrate tissue by agitating slides 10X each in EOTH series:
(1)50% (1) 75% (1) 95% (2)100%; Clear in 2 changes Xylene
MOUNTING MEDIA:
MICROSCOPE:
Permount with coverslip
Light microscope
NOTES:
REFERENCE:
Courtesy of Volker Nickeleit, The University of North Carolina at Chapel Hill, North
Carolina