Dec 07, 2023
Immunofluorescence Staining
- 1Department of Clinical and Movement Neurosciences, UCL Institute of Neurology, Queen Square, London WC1N 3BG, UK;
- 2Aligning Science Across Parkinson’s (ASAP) Collaborative Research Network, Chevy Chase, MD, 20 815, USA;
- 3Queen Square Brain Bank for Neurological Disorders (QSBB), 1 Wakefield Street, London WC1N 1PJ, UK
Protocol Citation: Toby J Curless, Hemanth Ramesh Nelvagal, Zane Jaunmuktane 2023. Immunofluorescence Staining. protocols.io https://dx.doi.org/10.17504/protocols.io.j8nlkowddv5r/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: August 07, 2023
Last Modified: May 31, 2024
Protocol Integer ID: 86049
Keywords: ASAPCRN
Funders Acknowledgement:
The Michael J. Fox Foundation for Parkinson’s Research (MJFF) and the Aligning Science Across Parkinson’s (ASAP) Initiative
Grant ID: ASAP-000478
Abstract
The protocol describes immunofluorescence staining on brain sections.
Materials
- 10% normal goat serum in PBS
- 100% Ethanol
- 70% Ethanol
- Antifademountant (P36980, Thermo Fisher Scientific)
- Coverslips
- DEPC H2O
- Epifluorescence microscope (Leica) Alexa 488 filters
- PBS
- Primary and secondary antibodies
- Steamer
- Xylene
Safety warnings
Breathing in xylene vapours in the air can cause irritation to the eyes, nose and throat and it can cause irritation, redness and swelling to the skin or eyes; and headaches, dizziness, vertigo and drowsiness. Please work to your institution approved health & safety policies, risk assessments and local procedures for the safe use, storage and disposal or xylene. Refer to the data safety sheet.
Preparation
Preparation
Generate tissue sections using standard microtome sectioning protocols.
Heat tissue dry tissue sections for 01:00:00 at 60 °C
1h
De-paraffinisation and Immunofluorescence
De-paraffinisation and Immunofluorescence
6h 6m 30s
Safety information
Please refer to the Xylene Data Safety Sheet and follow local approved risk assessements and protocols for safe handling and disposal of xylene.
De-paraffinise sections by 00:05:00 min washes in xylene (x3), 00:05:00 100 % ethanol (x1), 00:05:00 70 % ethanol (x1) and 00:05:00 DEPC H2O (x1).
20m
Rinse sections in PBS.
Encircle tissue with a hydrophobic ImmEdgeTM PAP pen (Vector laboratories) to contain solution.
Incubate tissue with H2O2for 00:15:00 mins at room temperature.
15m
Wash tissue 3x in PBS.
Antigen retrieval – heat 1 container (250 ml) DEPC H2O, and 1 container (250 ml) CC1 buffer to
99 °C using a steamer (BRAUN tribute collection).
Add slides to heated DEPC H2O for 10 s, then transfer to CC1 buffer and incubate at 99 °C for 00:15:00 .
15m
Remove slides and was 3x with PBS for 00:05:00 mins total.
5m
Block sections with 10 % normal goat serum in PBS for 02:00:00 .
2h
Wash tissue in PBS.
Incubate sections in primary antibodies (antibodies diluted in PBS) at 4 °C Overnight .
2h
Following overnight incubation, wash the sections in PBS 3x for a total of00:15:00 and shake off excess PBS after each 00:05:00 interval.
20m
Incubate sections with 1:100 secondary antibodies (e.g., Alexa Fluor 488-conjugated anti-mouse secondary antibody (Invitrogen) and Alexa Fluor 568-conjugated anti-rabbit secondary antibody (Invitrogen)) (for 00:30:00 mins at room temperature in the dark.
30m
Wash sections in PBS 3x for a total of 00:15:00 – whilst incubating, remove Antifade mountant (P36980, Thermo Fisher Scientific) from refrigerator and bring to room temperature.
15m
Counterstain sections with Hoechst (1:15000 (diluted in DEPC H2O)) for 00:00:30 sec
30s
4 °C Rinse sections in PBS 2x for a total of 00:06:00
6m
Coverslip slides using Antifade mountant (P36980, Thermo Fisher Scientific) and store slides in the dark at 4 °C .
Capture slides on an epifluorescence microscope (Leica) using Alexa 488 filters.