Mar 06, 2025
Immunohistochemistry Protocol (brain post-mortem tissue)
Forked from a private protocol
- Roger Barker1
- 1University of Cambridge
Protocol Citation: Roger Barker 2025. Immunohistochemistry Protocol (brain post-mortem tissue). protocols.io https://dx.doi.org/10.17504/protocols.io.n92ldroyog5b/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: March 06, 2025
Last Modified: March 06, 2025
Protocol Integer ID: 123911
Keywords: immunohistochemistry, antibody, sequential rehydration, counterstaining, ASAPCRN
Funders Acknowledgements:
Aligning Science Across Parkinson's (ASAP)
Grant ID: ASAP-00520
Aligning Science Across Parkinson's (ASAP)
Grant ID: ASAP-025170
Abstract
This protocol details the immunohistochemistry steps for antibody blocking over a period of 2 days.
Attachments
Materials
REAGENTS
- Xylene
- 70% EtOH
- 90% EtOH
- 100% EtOH
- dH2O
- Sodium citrate buffer: 2.94g Tri-sodium citrate (dehydrate), 1000ml Distilled Water (adjust to pH 6 with 1N HCl) then add 0.5ml Tween 20
- TBPS: 1x PBS + 0.05% Tween 20
- 3% H2O2 diluted in TPBS
- Blocking solution (e.g. 5% Normal horse serum in TPBS)
- Primary and Secondary Antibody
- ABC solution (DAB substrate): 5ml TPBS with 2 drops of reagent A and 2 drops of reagent B
- DAB stain: 5 ml dH20 with: 2 drops of buffer (pH 7.5), 4 drops of ABC and 2 drops of hydrogen peroxide
- Hematoxylin
- 1% Acid alcohol: 350ml industrial methylated spirit (denatured alcohol), 5ml HCl, 145ml dH2O
- Mounting medium: DPX
CONSUMABLES
- Slides
- Tip box for hematoxylin
EQUIPMENT
- Microwave
Safety warnings
For safety warnings and hazards, see the Safety Data Sheet (SDS).
Day 1
Day 1
5m
5m
Sequential rehydration
Xylene, 100% EtOH, 90% EtOH, 70% EtOH, dH2O – 5 min in each:
Rehydrate samples in Xylene for 00:05:00 .
5m
Rehydrate samples in 100% EtOH for 00:05:00 .
5m
Rehydrate samples in 90% EtOH for 00:05:00 .
5m
Rehydrate samples in 70% EtOH for 00:05:00 .
5m
Rehydrate samples in dH2O for 00:05:00 .
5m
Antigen Retrieval
Microwave slides at high power for 00:20:00 in 0.05 % (v/v) Tween 20 in Sodium Citrate Buffer , 6 .
Note
Sodium citrate recipe:
2.94 g Tri-sodium citrate (dehydrate)
1000 mL Distilled water
Mix to dissolve, adjust to 6 with 1N HCl and then add 0.5 mL Tween 20 and mix well.
*Store at RT for 3 months or at 4°C for longer storage.
20m
Wash
3 x 5 min with TBPS (0.05 % (v/v) Tween 20 in 1xPBS ):
Wash 1 of 3: 00:05:00 with TBPS (1x PBS + 0.05% Tween 20)
5m
Wash 2 of 3: 00:05:00 with TBPS (1x PBS + 0.05% Tween 20)
5m
Wash 3 of 3: 00:05:00 with TBPS (1x PBS + 0.05% Tween 20)
5m
Endogenous peroxidase blocking
Incubate slides in 3 % (v/v) hydrogen peroxide (H2O2) in TPBS at Room temperature for 00:15:00 .
15m
Wash
3 x 5 min with TBPS:
Wash 1 of 3: 00:05:00 with TBPS
5m
Wash 2 of 3: 00:05:00 with TBPS
5m
Wash 3 of 3: 00:05:00 with TBPS
5m
Blocking for 1 hour
Blocking Solution (e.g. 5% Normal horse serum in TPBS).
Primary antibody – Overnight incubation
Dilute in TPBS in appropriate concentration and incubate Overnight at 4 °C .
15m
Day 2
Day 2
4h 3m 31s
4h 3m 31s
Wash
3 x 5 min with TPBS:
Wash 1 of 3: 00:05:00 with TBPS
5m
Wash 2 of 3: 00:05:00 with TBPS
5m
Wash 3 of 3: 00:05:00 with TBPS
5m
Secondary antibody
Dilute antibody in blocking solution and incubate for 02:00:00 at Room temperature
2h
Wash
3 x 5 min with 1xTPBS:
Wash 1 of 3: 00:05:00 with 1xTBPS
5m
Wash 2 of 3: 00:05:00 with 1xTBPS
5m
Wash 3 of 3: 00:05:00 with 1xTBPS
5m
ABC solution (DAB substrate)
Note
*Prepare 00:30:00 before use!
Make 5 mL TPBS with 2 drops of reagent A and 2 drops of reagent B. Leave ABC on tissue for 01:00:00
1h
Wash
3 x 5 min with TPBS:
Wash 1 of 3: 00:05:00 with TBPS
5m
Wash 2 of 3: 00:05:00 with TBPS
5m
Wash 3 of 3: 00:05:00 with TBPS
5m
DAB Stain
Make 5 mL dH20 with: 2 drops of buffer (7.5 ), 4 drops of ABC and 2 drops of hydrogen peroxide. Incubate with DAB at Room temperature until colour change.
Wash
2 x 5 min with dH20:
Wash 1 of 2: 00:05:00 with dH20
5m
Wash 2 of 2: 00:05:00 with dH20
5m
Counterstaining with Hematoxylin
Place slides in tip box of hematoxylin for 00:00:30 .
30s
1 x 00:05:00 wash with dH20.
5m
Put slides in acid alcohol, 00:00:01 quickly in and out. Keep the slides in running water for 00:03:00 for blueing.
Note
1% Acid alcohol:
350 mL Industrial methylated spirit (denatured alcohol)
5 mL HCl
154 mL dH2O
3m 1s
Dehydration process
70% EtOH, 90% EtOH, 100% EtOH, Xylene:
Dehydrate samples in 70% EtOH.
Dehydrate samples in 90% EtOH.
Dehydrate samples in 100% EtOH.
Dehydrate samples in Xylene.
Mounting Slides
Mount slides using medium DPX.