How to Optimize mIHC Results: Antibody Diluents That Can't Be Ignored
Choosing the right primary antibody is critical in multiplex fluorescence immunohistochemistry (mIHC) experiments, but some small steps that are often overlooked, such as the selection of antibody diluents, can actually make the difference between success and failure. This article will explore how to optimize mIHC results by selecting the appropriate antibody diluent.
The importance of antibody diluents
Antibody diluents affect not only the specific interaction of the antibody with the target, but also the stability of the antibody. Commonly used diluents include PBST, TBST, PBS, and TBS. Understanding their differences is essential for precise selection.
Comparison of buffer compositions
|
Buffer |
Composition |
|
PBST |
Na2HPO4, KH2PO4, NaCl, KCl, Tween-20 |
|
PBS |
Na2HPO4, KH2PO4, NaCl, KCl |
|
TBS |
NaCl, KCl, Tris-base |
|
TBST |
NaCl, KCl, Tris-base, Tween-20 |
Difference Between PBS and TBS:
1) PBS: phosphate-based buffer system, with good pH stability, but may form precipitation with heavy metal ions, affecting some biochemical reactions.
2) TBS: A buffer system based on Tris-base, which has little interference to biochemical reactions, but the pH is easily affected by the environment and dilution factor.
Role of Tween-20 in IHC
Tween-20 is a non-ionic surfactant that increases the contact area between the antibody and the tissue, improves the refolding antigen ability of the antibody, and thus enhances specific binding.
Selection of antibody diluents
1) PBS: suitable for most experimental scenarios, including washing and culture.
2) TBS: It has little interference with antibodies and is suitable as an antibody diluent.
3) TBST: Contains Tween-20 and is suitable as an antibody diluent in mIHC experiments.
The role of blocking reagents
Blocking reagents, such as blocking serum from the same source as the secondary antibody or BSA, can reduce non-specific binding and increase the specificity of the antibody. If the secondary antibody species source of AbSon's multicolor kit is goat, then AbSon's goat serum (Cat. No. ABS933) can be used as a blocking agent.
conclusion
Choosing the right antibody diluent is critical to achieving perfect mIHC results. If your mIHC results are not satisfactory, it is recommended to try changing the antibody diluent to optimize the results.
Multicolor commonly used reagent recommendations:
|
classify |
Catalog number |
Product name |
specification |
|
fixed |
4% paraformaldehyde (general purpose tissue fixative solution) |
500mL |
|
|
Embedding (frozen samples) |
abs9756 |
OCT embedding medium |
110mL |
|
Antigen retrieval (paraffin sections) |
abs9342 |
Tris-EDTA antigen retrieval solution (10×, pH 9.0) |
100mL |
|
abs9248 |
Sodium citrate antigen retrieval solution (50×) |
100mL |
|
|
Antibody elution (samples that cannot be eluted with thermal repair, e.g., cells, frozen, bone tissue, etc.) |
Antibody eluent (for mIHC) |
30mL |
|
|
Cell permeabilization (required for intracellular indicators) |
abs9149 |
Quraton X-100 |
100mL |
|
Quenching endogenous peroxidases |
abs9333 |
Peroxidase blocking solution (H2O2 method) |
100mL |
|
Immunohistochemistry pen |
abs929 |
Immunohistochemistry pen plus: ABS9785 (waxy) |
1 stick |
|
Blocking (secondary antibody same source serum) |
Goat serum |
50mL |
|
|
abs9157 |
Bovine serum albumin |
100g |
|
|
Antibody diluents |
abs9299 |
Primary and secondary antibody diluents |
100mL |
|
Buffer |
abs952 |
TBST(10×) |
500mL×2 |
|
PBST(1×,pH7.4) |
500mL |
Absin provides antibodies, proteins, ELISA kits, cell culture, detection kits, and other research reagents. If you have any product needs, please contact us.
|
Absin Bioscience Inc. Email: worldwide@absin.net |
 Follow us on Facebook: Absin Bio |
