📋 Workflow
Full workflow, products add-to-cart directly1
Cell preparation
1 hSeed cells on coverslips or glass-bottom dishes, prepare at suitable confluency
💡 Tip Acid-wash coverslips to promote adhesion
⚠️ Caution Optimal density for clear observation
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2
Fix and permeabilize
30 minFix with 4% PFA, permeabilize with Triton X-100 to expose antigens
💡 Tip Long permeabilization unsuitable for membrane proteins
⚠️ Caution Over-permeabilization damages membrane
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3
Block and primary antibody
2 hBlock to reduce non-specific binding, incubate primary antibody overnight or 1-2 h
💡 Tip Include negative control (secondary only)
⚠️ Caution Optimize primary concentration
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4
Secondary and imaging
2 hIncubate fluorescent secondary, wash, mount with antifade+DAPI, image by fluorescence microscope
💡 Tip Incubate secondary in dark
⚠️ Caution Antifade mountant extends imaging window
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⚠️ 5 Common Beginner Mistakes
- ① Poor fixation/permeabilization causes false negatives or background
- ② Wrong permeabilization mislocalizes membrane proteins
- ③ Non-specific secondary binding elevates background
- ④ Photobleaching attenuates signal after imaging
- ⑤ Missing negative control weakens specificity judgment
❓ FAQ
+How to assess staining specificity?
Use secondary-only and peptide pre-adsorption controls; specific binding shows no signal.
+How to analyze co-localization?
Compute Pearson coefficient or 3D reconstruction.
+Can paraffin sections be used?
Yes, but requires dewaxing and antigen retrieval first.