📋 Workflow
Full workflow, products add-to-cart directly1
Fixation and permeabilization
30 minFix cells/sections with PFA, permeabilize with Triton X-100 to expose intracellular antigens
💡 Tip Different permeabilization for membrane vs intracellular
⚠️ Caution Over-permeabilization damages morphology
🧪 Materials for this step (click to shop by spec/brand)
2
Blocking
1 hBlock with BSA/serum-containing buffer to reduce non-specific binding
💡 Tip Adequate blocking lowers background
⚠️ Caution Keep blocking and dilution buffers consistent
🧪 Materials for this step (click to shop by spec/brand)
3
Primary antibody incubation
OvernightIncubate primary antibody against target, allow multiplex simultaneous labeling
💡 Tip Choose primary antibodies from different species for multiplex
⚠️ Caution Avoid cross-reactivity affecting co-localization
🧪 Materials for this step (click to shop by spec/brand)
4
Secondary antibody and imaging
2 hIncubate distinct fluorochrome secondaries, counterstain with DAPI, image by fluorescence/confocal
💡 Tip Match secondary to primary species
⚠️ Caution Protect from light to prevent quenching
🧪 Materials for this step (click to shop by spec/brand)
⚠️ 5 Common Beginner Mistakes
- ① Species mismatch gives no signal or cross-reactivity
- ② Insufficient blocking elevates background
- ③ Spectral overlap affects co-localization
- ④ Photobleaching attenuates signal over time
- ⑤ Missing negative control weakens specificity
❓ FAQ
+IF vs IHC?
IF uses fluorescence for live co-localization; IHC enzyme color suits morphology/archives.
+How to quantify?
Use confocal and image analysis to measure intensity, localization and co-localization coefficients.
+Notes for multiplex labeling?
Choose spectrally separated dyes, distinct-species primaries and single-color controls.