Aug 01, 2023
Immunohistochemistry (using floating section)
- Tae-Un Han1,
- Ellen Sidransky1
- 1National Institute of Health
Protocol Citation: Tae-Un Han, Ellen Sidransky 2023. Immunohistochemistry (using floating section). protocols.io https://dx.doi.org/10.17504/protocols.io.6qpvr36yovmk/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: July 28, 2023
Last Modified: May 31, 2024
Protocol Integer ID: 85658
Keywords: ASAPCRN
Funders Acknowledgement:
ASAP
Grant ID: ASAP-000458
Abstract
- This is an immunohistochemistry protocol used for evaluation of pathology in mouse brain.
- We successfully detected Gfap, Iba1, CD68, TH and alpha-Syn and phospho S129 alpha synuclein protein expression using this
protocol.
- In this protocol, floating sections are mounted on slides first before starting blocking step. In this way, large number of sections can
be stained more efficiently.
Materials
30 um thickness of floating tissue sections in PBS with 0.1% sodium azide.
- Washing: Glass Coplin jar
- PBST: PBS 1X + 0.05% Tween20
- Normal Goat Serum Blocking Solution:
2% goat serum (blocking)
1%BSA (stabilizer)
0.1% cold fish skin gelatin (blocking)
0.1% Triton X-100 (penetration enhancer)
0.05% Tween 20 (detergent and surface tension reducer)
0.05% sodium azide (preservative)
0.01M PBS, pH 7.2
Mix well and store at 4 ºC for up to 6 months.
- Primary Antibody Dilution Buffer
1%BSA (blocking & stabilizer)
0.1% cold fish skin gelatin (blocking)
0.5% Triton X-100 (penetration enhancer)
0.05% sodium azide (preservative)
0.01M PBS, pH 7.2-7.4
Mix well and store at 4 ºC. up to 6 months.
- Secondary Antibody Dilution Buffer:
0.01M PBS, pH 7.2
0.05% Tween 20
- List of antibodies used for neuropathology evaluation with IHC
| A | B | C | D | |
| Antibodies | Cat# | Company | dilution | |
| Anti-Synuclein α Antibody | AB5038 | Sigma-Aldrich | 1:1000 | |
| Recombinant Anti-Alpha-synuclein (phospho S129) antibody [EP1536Y] | ab51253 | Abcam | 1:1000 | |
| GFAP Antibody , Rabbit Polyclonal | 16825-1-AP | Proteintech | 1:4000 | |
| Recombinant Anti-Iba1 antibody [EPR16588] | ab178846 | Abcam | 1:1000 | |
| Anti-Tyrosine Hydroxylase | AB152 | Sigma-Aldrich | 1:1000 | |
| Anti-CD68 antibody | ab125212 | Abcam | 1:1000 | |
| Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) : 2nd antibody | ab150077 | Abcam | 1:1000 |
Mount floating sections onto slides and dry for 00:30:00 (<45 min), While section drying, PAP pen drawing line and dry 00:05:00 .
35m
PBS rehydrate 00:10:00 , Wash with PBS for 00:05:00 three times.
15m
Blocking sol 500 µL , incubate 01:00:00 at Room temperature .
1h
Replace buffer to primary antibody sol, incubate Overnight at 4 °C .
10m
Wash in PBST three times for 00:05:00 , 00:10:00 and 00:05:00 .
20m
Secondary antibody sol, incubate for 01:30:00 at Room temperature .
1h 30m
Wash in PBS three times for 00:05:00 , 00:10:00 and 00:05:00 .
20m
Mounting with Vectorshield mounting media