Nov 13, 2025
4-HNE immunofluorescence staining
- lene.devos 1,
- tine.wylin 1
- 1Abdominal Transplantation, KU Leuven
- Abdominal Transplantation - KUL
Protocol Citation: lene.devos , tine.wylin 2025. 4-HNE immunofluorescence staining . protocols.io https://dx.doi.org/10.17504/protocols.io.8epv5k56jv1b/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working in rat liver and kidney tissue as well as pig liver and lung tissue.
Created: October 29, 2025
Last Modified: November 13, 2025
Protocol Integer ID: 231005
Keywords: 4-hydroxynonenal, lipid peroxidation, immunofluorescence, liver, kidney, lung, rat, pig, hne immunofluorescence, hydroxynonenal, immunofluorescence, hne, using ab46545
Abstract
This protocol provides an overview of the different steps involved in immunofluorescence staining of 4-hydroxynonenal (4-HNE) using ab46545. This protocol has proven to be effective in rat kidney and liver tissue as well as pig liver and lung tissue.
Materials
- Paraffin tissue section of 5 μm
- Coplin jars (a volume of 250 ml is enough to immerse slides)
- Easy DipTM container + staining rack
- Xylene
- Ethanol (50% - 100%)
- MiliQ (MQ)
- Citric acid (0,1 M):
| A | B | C | D | |
| Reagent (concentration) | Volume needed | |||
| Citric acid (anhydrous powder) (192,123 g/mol) | 9,60 g | 4,80 g | 1,92 g | |
| MQ | 500 ml | 250 ml | 100 ml | |
- Sodium citrate (0,1 M, pH 6):
| A | B | C | D | |
| Reagent (concentration) | Volume needed | |||
| Sodium citrate (powder) (258,07 g/mol) | 12,91 g | 6,46 g | 2,58 g | |
| MQ | 500 ml | 250 ml | 100 ml | |
Note: Start with a lower volume and adjust to pH. Afterwards, you can fill until the desired volume. Check final pH to be sure.
- Citric acid buffer:
| A | B | |
| Reagent (concentration) | Volume needed | |
| Citric acid (0,1M) | 2,30 ml | |
| Sodium citrate (0,1M, pH 6) | 10,70 ml | |
| MQ | Add until total volume of 130 ml |
- Microwave
- Slide container with wet tissues
- Tris NaCl buffer:
| A | B | |
| Reagent (concentration) | Volume needed | |
| Tris-HCl (0,1 M, pH 7,5) (157,6 g/mol) | 7,88 g | |
| NaCl (0,15 M) (58,44 g/mol) | 4,38 g | |
| MQ | 500 ml |
- TNT (Tris NaCl, Tween20) buffer:
| A | B | |
| Reagent (concentration) | Volume needed | |
| Tris NaCl buffer | 50 ml | |
| Tween20 (0,05%) | 25 µl |
- TNB (Tris NaCl Blocking) buffer:
| A | B | |
| Reagent (concentration) | Volume needed | |
| Tris NaCl buffer | 50 ml | |
| Bovine Serum Albumin (0,5%) | 0,25 g |
- Goat serum
- Primary antibody: Rabbit Polyclonal 4-HNE antibody (1/500; Cat.# ab46545; RRID: AB_722490)
- Secondary antibody: Goat anti-Rabbit IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor‱ 488 (4 µg/ml; Cat.# A-11008; RRID: AB_143165)
- DAPI (Cat.#62248)
- PBS (1x)
- Coverslips
- Slow Fade Diamond antifade (Fisher Scientific (15,441,244))
- Nail polish
Troubleshooting
Problem
Insufficient antigen retrieval
Solution
Microwave settings might differ between brands/types. Test at which wattage and time combination the antigen retrieval buffer reaches boiling point, without buffer overflowing. Keeping the lid open can help to prevent boiling over. Use this setting to preheat the buffer. Next, find a setting that helps maintain this temperature without the buffer boiling over. This setting can be used for the slides.
Before start
Prepare all buffers and reagents before start of deparaffinization
Deparaffinization
25m
Submerge the slides in the following reagents:
5’ Xylene
5’ Xylene
3’ 100% Ethanol
3’ 90% Ethanol
3’ 70% Ethanol
3’ 50% Ethanol
3’ MQ
25m
Antigen retrieval
42m 30s
Fill the Easy DipTM container with citric acid buffer (±120-130 ml)
Preheat the citric acid buffer in the microwave for 1,5’ at 750W (keep the lid of the container open).
1m 30s
Immerse the slides in the preheated buffer
Place the slides in the microwave for 2 cycles of 4,5’ at 90W with 1-2 minutes in between (keep the lid of the rack elevated).
11m
Let the slides cool down for 30’ in the buffer
30m
Incubation with primary antibody
1d 1h 5m
Prepare a chamber (slide container) with wet tissues on the bottom to maintain humidity
Wash 5’ with TNT
5m
Dry slides carefully with tissue paper without affection the sections
Block for 1h with TNB + 1/50 normal goat serum
1h
Incubate the slides with 1st antibody (TNB + 1/50 normal goat serum + 1/500 4HNE primary antibody) overnight at 4°C
1d
Incubation with secondary antibody
1h 15m
Wash 3x 5’ with TNT
15m
Dry slides carefully with tissue paper
Cover the chamber from light
Incubate the slides for 1h with 2nd antibody (TNB + 1/50 normal goat serum + 4 µg/ml 2nd
antibody) at room temperature
1h
DAPI nuclear staining
35m
Wash 3x 5’ with TNT
15m
Dry slides with tissue paper
Add DAPI (1/2000 in PBS) for 5’
5m
Dry slides with tissue paper
Add DAPI again (1/2000 in PBS) for 5’
5m
Wash 5’ PBS at RT in glass jar or Easy DipTM container
5m
Repeat wash 5’ PBS at RT in glass jar or Easy DipTM container
5m
Mounting
Dry slides with tissue paper
Add 1 drop of slow fade in the middle
Place a coverslip on top of the slide and remove air bubbles
Apply nail polish on the borders to secure the coverslip
Store overnight in the dark at room temperature
After drying/visualising, the slides can be stored in the fridge or at -20°C
Acknowledgements
We would like to thank Sai Kocherlakota, PhD for sharing his expertise in this staining.