[Histonet] Problems with DIF staining of C4d

[Morken, Timothy]

Beth, which C4d are you using? And it is a FITC-labeled primary?



We use it as a two-step rather than FITC-labeled primary. That increases the sensitivity significantly. Gloms have C4d so act as an internal control.



We use
C4d antibody, 100ul vial, unlabeled primary.  Diluted 1:200 in Dako or Bond diluent.
Quidel, Cat#  A-213

Secondary Ab:
Antibody, Goat anti Mouse IgG H+L FITC 1.5mg. Diluted 1:120 in Dako or Bond diluent.
Jackson Immuno Research
115-095-062


We store the concentrate in the fridge. We use it daily so go through it pretty quickly, but we have it validated for two years at 2-8C. We make fresh dilutions a couple times per week.



We stain on the Bond Autostainer, but manual will work fine as well.





Tim Morken

Supervisor, Electron Microscopy/Neuromuscular Special Studies

Department of Pathology

UC San Francisco Medical Center



-----Original Message-----
From: O'Neil, Beth A. via Histonet <histonet at lists.utsouthwestern.edu>
Sent: Tuesday, June 08, 2021 10:32 AM
To: Histonet <histonet at lists.utsouthwestern.edu>
Subject: [Histonet] Problems with DIF staining of C4d



We perform manual DIF staining at our facility and C4d has always been a problem.  We end up repeating the stain more times than not due to lack of staining.  We aliquot the FITC and freeze at -70C until ready for use.  We also started doing the same with the C4d.  Sometimes it works and sometimes it doesn't.  My pathologist said it is not the FITC but the C4d itself.  I have contacted the manufacturer of my C4d but they are very slow to respond.  Any suggestions or experiences would be appreciated.



Beth ONeil

WVU Medicine, JW Ruby Memorial Hospital



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