Although the causes of non-specific background staining in immunohistochemistry (IHC) and immunofluorescence (IF) applications may differ slightly, they can obscure the detection of your specific signal. Learn about the sources of background staining and how to troubleshoot unwanted background in IHC and IF applications.
Topics of discussion include:
- Why background staining is problematic
- How to identify sources of background
- Strategies used to eliminate background
If you’re in a hurry, time references have been added to help you skip to sections of interest from this overview:
Introduction 1.11
What is background? 1.30
Why is background in IHC & IF an issue? 3.19
How to trouble shoot IHC & IF 4.20
Background sources from tissue 4.45
IHC workflow 6.11
Identifying the source of background 7.12
HRP Quenching methods 8.30
AP Quenching methods 9.11
2nd Deletion Control 9.50
Species cross reactivity caused background 11.01
Inadequate blocking for detection reagents 13.19
The importance of buffer washes 14.26
Other contributing factors 15.13
Second deletion control for ABC methods 15.42
Checking the complete staining system 16.25
Immunofluorescence troubleshooting 18.20
Tissue autofluorescence 19.42
Methods of blocking autofluorescence 20.29
Lipofuscin based autofluorescence 21.00
Further tools & Resources 22.30