TL;DR: For a common IHC heat-induced epitope retrieval buffer, prepare 10 mM sodium citrate at pH 6.0. Using trisodium citrate dihydrate, use 2.94 g per litre. Tween 20 at 0.05% is optional if validated for the antibody and tissue.
Citrate buffer pH 6 is one of the most established heat-induced epitope retrieval (HIER) buffers for formalin-fixed paraffin-embedded tissue. Heating can reverse or loosen some formaldehyde-induced crosslink effects and improve access of antibodies to masked epitopes.
10 mM citrate buffer pH 6 recipe
| Final volume | Trisodium citrate dihydrate | Optional Tween 20 (0.05%) |
|---|---|---|
| 100 mL | 0.294 g | 50 µL |
| 500 mL | 1.47 g | 250 µL |
| 1 L | 2.94 g | 500 µL |
Preparation
- Dissolve trisodium citrate dihydrate in approximately 80–90% of the final water volume.
- Measure pH at room temperature.
- Adjust carefully to pH 6.0 with HCl.
- Add Tween 20 only if the validated protocol uses it.
- Bring to final volume with purified water.
How citrate antigen retrieval works
Formalin fixation creates protein crosslinks that can mask antibody epitopes. Heating fixed sections in retrieval buffer can restore accessibility for many targets. The original microwave antigen-retrieval studies showed substantial improvement in immunostaining for many antigens, but not all antibodies respond equally.
Typical HIER workflow
- Deparaffinise and rehydrate tissue sections.
- Preheat citrate buffer in the chosen retrieval system.
- Heat sections in buffer using a pressure cooker, water bath, steamer or microwave under validated conditions.
- Allow slides to cool gradually in the retrieval buffer.
- Wash and continue with blocking and immunostaining.
Do not treat the heating time above as universal. Retrieval intensity depends on tissue, fixation, antibody and heating device.
Citrate pH 6 vs Tris-EDTA pH 9
Some epitopes retrieve best at acidic pH, others at alkaline pH. If an antibody performs poorly in citrate pH 6, test an alkaline Tris-EDTA retrieval system rather than simply extending heating indefinitely.
Common mistakes
- Letting slides dry during retrieval or cooling.
- Changing retrieval pH without revalidating the antibody.
- Overheating tissue until morphology is damaged.
- Assuming one retrieval buffer works for every target.
Troubleshooting
| Problem | Likely cause | Fix |
|---|---|---|
| Weak staining | Epitope insufficiently retrieved | Optimise heat exposure or compare alkaline retrieval buffer. |
| Tissue damage | Over-retrieval | Reduce temperature/time and confirm slide adhesion. |
| High background | Over-retrieval or antibody concentration too high | Reduce retrieval intensity and re-titrate antibody. |
Frequently asked questions
Can I reuse citrate retrieval buffer?
For reproducibility, fresh or clearly validated reused buffer is preferable. Repeated heating can change volume and concentration through evaporation.
Should I add Tween 20?
It is optional. Some laboratories include 0.05% Tween 20; others use citrate alone. Keep the condition that has been validated for the antibody and tissue.
Related ABMIUM guides
Primary research references
- Shi SR, Key ME, Kalra KL. Antigen retrieval in formalin-fixed paraffin-embedded tissues by microwave heating. J Histochem Cytochem. 1991.
- Shi SR et al. Citrate buffer or urea antigen retrieval for androgen receptor immunohistochemistry. J Histochem Cytochem. 1993.
- Utility of microwave-citrate antigen retrieval in diagnostic immunohistochemistry. 1995.