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	abs9179	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.)	abs994	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)	abs933	Goat serum	50mL
	abs9157	Bovine serum albumin	100g
Antibody diluents	abs9299	Primary and secondary antibody diluents	100mL
Buffer	abs952	TBST(10×)	500mL×2
	abs9340	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