Sep 11, 2025
Immunhistochemistry (Stadelmann Lab - single antibody, AP)
- Jonas Franz1,
- Katja Schulz1,
- Christine Stadelmann1
- 1University Medical Center Göttingen, Neuropathology
Protocol Citation: Jonas Franz, Katja Schulz, Christine Stadelmann 2025. Immunhistochemistry (Stadelmann Lab - single antibody, AP). protocols.io https://dx.doi.org/10.17504/protocols.io.3byl4952rgo5/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
Created: June 28, 2024
Last Modified: September 11, 2025
Protocol Integer ID: 102565
Keywords: ASAPCRN, alkaline phosphatase secondary antibody, ffpe human brain tissue, human brain tissue, using alkaline phosphatase, secondary antibody, single antibody, immunhistochemistry
Abstract
Staining of FFPE human brain tissue using alkaline phosphatase secondary antibodies.
Troubleshooting
Paraffin removal and rehydration
Immunohistochemistry was performed on 2-3 µm thick paraffin sections.
Remove Excess Paraffin at 56° for 1h-24h
1h
Rehydration
4x Xylol à 5 min
20m
1x Isopropanol:Xylol 1:1 à 3 min
3m
2x 100% Isopropanol à 3 min
6m
1x 90% Isopropanol à 3 min
3m
1x 70% Isopropanol à 3 min
3m
1x 50% Isopropanol à 3 min
3m
3x wash in aqua dest
1m 30s
Antigen Retrieval
Antigen Retrieval
heat antigen retrieval (1x citrate buffer 10 mM , pH 6) - 30 min steamer
30m
3x wash TBS
1m 30s
formic acid (98%) (for Amyloid beta immunhistochemistry) - 1minute at RT
1m
3x wash TBS
1m 30s
Blocking
15h 36m 10s
3% hydrogen peroxide in TBS 10 min @ RT
10m
3x wash TBS
1m 30s
Blocking in 10% normal goat serum in TBS for 10 min @ RT
10m
Decant 10% NGS in TBS and move directly to the next step without washing
Staining
15h 36m 10s
Incubation of primary antibodies over night with respective dilution in 10% NGS @RT in a humid chamber
| target antigen - human | host species | Clone | Company | Catalog Number | Dilution | |
| beta-Amyloid | mouse | 6E10 | Zytomed Systems GmbH | Z 932002-Y | 1:500 |
12h
3x wash with TBS
1m 30s
Incubation with secondary antibody coupled to alkaline phospohatase 1:100 at RT for 2h in a humid chamber
| target antigen | host species | Clone | Company | Catalog Number | Dilution | |
| mouse IgG | goat | Jackson Immuno Research | 115-055-003 | 1:100 |
2h
3x wash TBS
1m 30s
Slides were developed using FastBlue 30-60 min
49 mL TBS pH 7.5, 1 mL Naphtol AS Mix Phosphat, 50 µL Levamisol, 25 mg FastBlue
-> Filtration
1h
3x wash TBS
1m 30s
Counterstaining + Mounting
15h 36m 10s
Hematoxylin 10s
10s
10 min tap water
10m
Aquatex mounting