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
Sent: Tuesday, June 08, 2021 10:32 AM
To: Histonet
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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