📋 Workflow
Full workflow, products add-to-cart directly1
Sectioning and dewaxing
1 hBake paraffin sections, deparaffinize in xylene, rehydrate through ethanol gradient
💡 Tip Complete deparaffinization is essential
⚠️ Caution Consistent thickness and flat sections
🧪 Materials for this step (click to shop by spec/brand)
2
Antigen retrieval
30 minExpose masked antigens by heat-induced (citrate/EDTA buffer) or enzymatic retrieval
💡 Tip Optimize per antibody datasheet
⚠️ Caution Excess heat/time damages tissue or antigen
🧪 Materials for this step (click to shop by spec/brand)
3
Block and primary antibody
OvernightBlock endogenous peroxidase and protein sites, incubate with primary antibody
💡 Tip Include negative and isotype controls
⚠️ Caution Optimize primary antibody concentration
🧪 Materials for this step (click to shop by spec/brand)
4
Secondary, develop and counterstain
2 hIncubate HRP-labeled secondary, develop with DAB substrate, counterstain with hematoxylin and mount
💡 Tip Control DAB time to avoid over-development
⚠️ Caution Stop at optimal color by washing
🧪 Materials for this step (click to shop by spec/brand)
⚠️ 5 Common Beginner Mistakes
- ① Poor dewax/retrieval causes weak signal or high background
- ② Unblocked endogenous peroxidase darkens background
- ③ Wrong antibody concentration gives non-specific or missed staining
- ④ Over-developed DAB obscures staining
- ⑤ Missing controls hinder specificity judgment
❓ FAQ
+Heat vs enzymatic retrieval?
Heat is preferred for most antibodies; choose by datasheet when unverified.
+How to assess specificity?
Use negative, isotype and pre-adsorption controls; specific signal shows expected localization.
+High background?
Strengthen blocking, lower antibody, longer washes or shorter development.