Non-specific staining is often seen in erythrocytes, granulocytes, monocytes, and in muscle. This is due to incompletely-blocked endogenous peroxidase. Spleen showing typical, non-specific staining of erythrocytes due to incomplete blocking of endogenous peroxidase. Here the natural peroxidase pres...
When running immunohistochemistry (IHC) experiments, potential IHC staining issues may occur. This video aims to address common issues: Weak or No Staining (0:03) High Background/Non-specific Staining (1:04) Sections Floating Away/Coming Loose (2:05)...
Preventing Non-specific Staining Primary Antibody Selection & Optimization Protocol for Heat-Induced Epitope Retrieval (HIER) Protocol for Making a 4% Formaldehyde Solution in PBS Protocol for the Preparation & Fixation of Cells on Coverslips
These will help to account for the majority of non-specific staining and any autofluorescence that you might have in your sample.As stated, always run your controls alongside your main experiment. If you have to perform multiple rounds of staining due to space constraints in your experimental ...
the duration and other fixation conditions should be carefully optimized as they can impact preservation and tissue integrity. For instance, overfixation with formaldehyde can mask the epitope and produce strong non-specific staining, while underfixation may reduce or abolish tissue immunoreactivity. Follo...
If you're pushed for reagents, space in your staining setup, or have limitations due to precious tissue, the two controls that you must always run are the endogenous and secondary only controls. These will help to account for the majority of non-specific staining and any autofluorescence that...
Immunohistochemistry (IHC) is a technique that uses an antibody to bind a specific antigen in a tissue section and is visualized with a fluorophore or colored substrate. Locations of stained proteins helps us understand cell types and their functions within a tissue. Basic IHC staining typically re...
Learn more about Immunohistochemistry (IHC) protocol for preparation and immunofluorescence staining, including tissue fixation and freezing. (142)
A Spleen showing typical, non‑specific staining of erythrocytes due to incomplete blocking of endogenous peroxidase. Here the natural peroxidase present in the red cells has reacted with the DAB chromogen. TABLE OF CONTENTS / 43 44 / GUIDE TO BETTER IHC PROTEIN BLOCK NEEDED • Positive...
A negative tissue control (tissue or cells known to lack expression of your target antigen) should be run with each immunohistochemistry experiment to determine the amount of background staining. The presence of non-specific staining will be associated with the deposition of chromogenic or fluorescent...