Incorrect Antibody Diluent Selection

Symptom

Suboptimal signal intensity or background issues despite correct antibody concentration. Different antibodies show dramatically different performance in TBST/5% NGS versus SignalStain Antibody Diluent.

1. 1 Using TBST/5% Normal Goat Serum when SignalStain Antibody Diluent is recommended for specific antibody
2. 2 Using SignalStain Antibody Diluent when TBST/5% NGS is optimal for specific antibody
3. 3 Antibody concentration not re-titrated after switching to non-recommended diluent

1. 1 Always use recommended primary antibody diluent as specified on product datasheet (e.g., Phospho-Akt #4060 requires SignalStain Diluent; Phospho-EGFR #4407 requires TBST/5% NGS)
2. 2 If using alternative diluent, perform complete antibody titration to optimize concentration
3. 3 Check product datasheet and product page for specific diluent recommendations before starting experiment

Related Video (3)

Cell Signaling Technology ★ 95

Immunohistochemistry Protocol for Paraffin embedded Tissue Sections

"Official CST IHC protocol video directly addressing the technique and likely covering antibody diluent selection best practices"

Bilibili (China-Accessible Mirrors) ★ 92

Immunohistochemistry on Paraffin-Embedded Sections (CST Demo)

"CST-authored step-by-step IHC demonstration on paraffin sections that would show proper antibody dilution and diluent application techniques"

Cell Signaling Technology ★ 78

Better IHC Step 1: Antigen Retrieval

"Focused optimization series on IHC troubleshooting including antigen retrieval and staining quality, contextualizing where diluent choice impacts results"