Jun 18, 2024
Version 1
Fixation and immunocytochemistry (fluorescence) in PFF-treated cultures V.1
- 1Yale University;
- 2Tufts University
Protocol Citation: Maria Iuliano 2024. Fixation and immunocytochemistry (fluorescence) in PFF-treated cultures. protocols.io https://dx.doi.org/10.17504/protocols.io.x54v922q4l3e/v1Version created by Maria Iuliano
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 06, 2024
Last Modified: June 18, 2024
Protocol Integer ID: 101845
Keywords: ASAPCRN
Funders Acknowledgement:
ASAP
Grant ID: 020616
Disclaimer
DISCLAIMER – FOR INFORMATIONAL PURPOSES ONLY; USE AT YOUR OWN RISK
The protocol content here is for informational purposes only and does not constitute legal, medical, clinical, or safety advice, or otherwise; content added to protocols.io is not peer reviewed and may not have undergone a formal approval of any kind. Information presented in this protocol should not substitute for independent professional judgment, advice, diagnosis, or treatment. Any action you take or refrain from taking using or relying upon the information presented here is strictly at your own risk. You agree that neither the Company nor any of the authors, contributors, administrators, or anyone else associated with protocols.io, can be held responsible for your use of the information contained in or linked to this protocol or any of our Sites/Apps and Services.
Abstract
This protocol outlines methods used for the preparation of in vitro models for fluorescent imaging including primary rat and mouse hippocampal cultures treated with PFFs.
Image Attribution
Maria Iuliano -60x image of rat hippocampal neurons (DIV28) after 7-day treatment with PFFs. Map2 (Blue) and phopsho-a-syn Ser129 (green)
Guidelines
Parameters used in this protocol may be optimized and modified for individual use depending on experimental needs.
Materials
1% SDS for PFF inactivation
4% PFA 4% sucrose in 1x PBS
1x PBS
ICC Buffer: 5% NGS in 1x PBS
ICC Blocking Buffer: 5% NGS 0.3% Triton-X in 1x PBS\
Primary & secondary antibodies of choice
Microscope slides
Forceps
Mounting medium of choice
Protocol materials
Aqua-Poly/MountPolysciences, Inc.Catalog #18606-100
Step 9
Safety warnings
When working with PFFs, it is best to follow proper safety precautions and wear appropriate PPE, including lab coat, gloves, sleeve guards, safety glasses or face shield, and N95 or FFP2 mask.
Inactivation and disposal of PFFs and PFF-contaminated items can be done using 1% SDS as described in literature.
Collect and dilute solutions into 10vol 1% SDS for 01:00:00 at Room temperature
Discard used tips into primary collection container with 1% SDS
Inactivated liquid can be discarded with liquid waste
Discard inactivated items in primary container into biohazard
Wipe surfaces or contaminated tools down completely with 1% SDS, followed by water, then EtOH
CITATION
Before start
Please read safety guidelines ahead of working with PFFs
Fixation and Immunocytochemistry (Fluorescence)
Fixation and Immunocytochemistry (Fluorescence)
4h 30m
In a cell culture hood remove media and wash briefly with 1x PBS.
Safety information
Collect media and first wash as PFF waste for inactivation. Once in fixative, plate(s) can be moved to benchtop.
2m
20 rpm, Room temperature , 00:15:00 Fix using 4% PFA 4% sucrose in 1xPBS with gentle rocking/shaking.
Note
Check whether the antibodies you intend to use require specific fixation. Optimization may be needed.
15m
20-30 rpm, Room temperature , 00:10:00 Wash 1x PBS. Repeat 3 times.
Note
Pause Point: Coverslips can be stored after fixation for up to 2 weeks (or maybe more), though immediate processing is ideal. Store at 4C away from light, with the plate wrapped in parafilm to reduce evaporation.
30m
20-30 rpm, Room temperature , 00:45:00 Block in blocking buffer (5% NGS, 0.3% Triton-X in 1x PBS)
45m
20 rpm, 4°C Overnight Incubate in solution containing primary antibodies diluted in 5% NGS in 1x PBS
Note
Antibodies
| A | B | C | |
| Antibody | Company | Catalog # | |
| Gephyrin | Synaptic Systems | 147111 | |
| HA (HA-tag) | BioLegend | 901513 | |
| HA (HA-tag) | Cell Signaling | 3724 | |
| Homer | Synaptic Systems | 160003 | |
| MAP2 | Millipore | AB5543 | |
| MAP2 | Millipore | MAB364 | |
| Neurofilament | Millipore | AB5539 | |
| Phospho-α-syn (Ser129) | Biolegend | 825701 | |
| VGAT | Synaptic Systems | 131003 | |
| VGLUT1 | Millipore | AB5905 |
Some commonly used antibodies in our lab for PFF-treated cultures. Dilutions may need to be optimized to individual needs.
20-30 rpm, Room temperature , 00:10:00 Wash 1x PBS. Repeat 3 times.
30m
20 rpm, 4°C, 02:00:00 Incubate in solution containing secondary antibodies diluted in 5% NGS in 1x PBS
Note
Secondary incubation can vary (up to O/N), but typically 2-3hr at 4C works well. 1hr at RT can also work, though it may result in excess background signal, depending on the antibody
Typically, we use Alexa Fluor secondaries from Life Technologies, except for anti-chicken CF 405 (Millipore SAB4600466).
2h
20-30 rpm, Room temperature , 00:10:00 Wash 1x PBS. Repeat 3 times.
30m
Mount coverslips onto microscope slides using Aqua-Poly/MountPolysciences, Inc.Catalog #18606-100 or preferred mounting medium.
5m
Let cure 48:00:00 before imaging
2d
Citations
Bousset L, Brundin P, Böckmann A, Meier B, Melki R. An Efficient Procedure for Removal and Inactivation of Alpha-Synuclein Assemblies from Laboratory Materials.
https://doi.org/10.3233/JPD-150691