Jan 23, 2025
Immunofluorescence on acute hippocampal slices following electrophysiology
- Quinn Pauli1,
- Robert Bonin1
- 1Leslie Dan Faculty of Pharmacy, University of Toronto, Toronto, Ontario, Canada
Protocol Citation: Quinn Pauli, Robert Bonin 2025. Immunofluorescence on acute hippocampal slices following electrophysiology . protocols.io https://dx.doi.org/10.17504/protocols.io.4r3l2923qv1y/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: December 05, 2024
Last Modified: January 23, 2025
Protocol Integer ID: 114319
Keywords: Immunofluorescence , Staining , Hippocampal slices, Electrophysiology, IF
Funders Acknowledgements:
Natural Sciences and Engineering Research Council of Canada
Grant ID: RGPIN-2024-06215
Abstract
This protocol describes protein detection using immunofluorescence on hippocampal slices obtained from adult mice following electrophysiology recordings. Here, we detail the fixation, cryopreservation, slicing and staining methods to produce reliable detection of fluorescently labeled proteins. It is intended to enable spatial quantification of proteins (e.g. glutamate receptors) following electrophysiology recordings.
Materials
Reagents:
Phosphate-buffered saline (PBS)
Paraformaldehyde 4% (in PBS)
Triton-X
Horse serum (or other blocking agent)
Sucrose
Optimal cutting temperature compound (OCT)
Dry ice
Mountant (e.g. ProLong‱ Glass Antifade, ThermoFischer Scientific P36980)
Equipment:
Cryostat, chucks and blades
Microscope slides
Culture dishes
Pasteur pipettes
Cryomolds
Parafilm
Tissue spatula
Tweezers
PAP pen
Coverslips
Clear nail polish
Safety warnings
If you choose to use this protocol, you do so at your own risk and must ensure that any local guidance is adhered to.
Ethics statement
Experiments involving animals must be conducted according to internationally-accepted standards and should always have prior approval from an Institutional Animal Care and Use Committee (IACUC) or equivalent ethics committee(s). Animal care and experimental procedures were reviewed and approved by the
Animal Ethics and Compliance Program at the University of Toronto and conducted
in accordance with the Canadian Council on Animal Care (CACC) guidelines.
Fixation
Fixation
1w 0d 2h 15m
1w 0d 2h 15m
Immediately after electrophysiology recording is complete, transfer hippocampal slice from recording solution to 2-3 mL fixative solution (4% PFA in PBS) in a small culture dish using a pasteur pipette. Leave on rocker at Room temperature for 02:00:00
2h
Wash 3 x 00:05:00 with 1 X PBS
15m
Replace PBS with 30% sucrose solution (w/v in PBS) and seal culture dish with parafilm
Store at 4 °C for 1-2 weeks
1w
Cryo-embedding
Cryo-embedding
5m
5m
Transfer slice to a cryomold, placing it flat at the bottom using a spatula. Include orientation markers on outside of mold
Using a rolled kimwipe, soak up any excess sucrose solution without directly touching the slice
Pour Optimal Cutting Temperature compound (OCT) overtop of slice and fill the mold, being careful not to displace the slice. Remove any bubbles in the OCT carefully using a pair of tweezers or spatula
Embed cryomold in crushed dry ice to flash freeze
5m
When frozen, wrap cryomold in parafilm and/or a small plastic bag, label and store at -80 °C until processing
Cryo-sectioning
Cryo-sectioning
2h
2h
Place sample and tools in cryostat for 00:30:00 to acclimatize to -22 °C
30m
Pour OCT on specimen chuck and allow it to freeze. When nearly frozen, place a weight on top to flatten surface of OCT
Remove OCT-embedded sample from cryomold. Place a small amount of OCT on flat surface of chuck and immediately place sample flat on top, with the tissue facing upwards.
Tighten the chuck in the chuck holder, orienting the tissue with the longest side facing the blade
Adjust the chuck holder knobs such that the OCT is flush with the blade before slicing
Slice 20 µm sections, ensuring that the blade remains flush with the sample (not on an angle). Adjust as needed before proceeding.
Collect each slice from under the anti roll plate and orient it appropriately. Pick slice up swiftly with a Room temperature microscope slide
Collect 16-20 slices across 4-5 microscope slides and allow slides to dry for 01:00:00 at Room temperature
1h
Store slides at -20 °C until staining
Immunostaining - Primary Ab Incubation
Immunostaining - Primary Ab Incubation
2h 30m
2h 30m
Allow slides to acclimatize to Room temperature for 01:00:00
1h
Use a hydrophobic barrier PAP pen to outline sections on microscope slide and allow to dry
5m
Gently wash slides 5 x 00:05:00 with 0.1% Triton-X in PBS (PBS-T) using a pasteur pipette (~250 µL /slide)
25m
Incubate in blocking solution (10% horse serum in PBS-T) for 01:00:00 at Room temperature
1h
Incubate in primary antibody cocktail (typically 1:1000 dilution) in blocking solution overnight at 4 °C
Immunostaining - Secondary Ab Incubation
Immunostaining - Secondary Ab Incubation
4h 20m
4h 20m
Wash slides 5 x 00:05:00 with PBS-T
25m
Incubate in secondary antibody cocktail (typically 1:500 dilution) in blocking solution for 02:00:00 at Room temperature protected from light
2h
Wash slides 5 x 00:05:00 with PBS-T, keeping slides protected from light in between washes
25m
Incubate in DAPI (1:500 in PBS) for 00:10:00 at Room temperature protected from light
10m
Wash slides 2 x 00:05:00 in PBS, keeping slides protected from light in between washes
10m
Let slides dry 00:10:00 on an angle protected from light
10m
Mount slides with an appropriate mounting medium and coverslip
Wait 00:30:00 for mountant to solidify
30m
Seal edges of coverslip with nail polish to prevent from drying out and allow to dry
30m
Store in fridge until imaging
Imaging
Imaging
Clean slides with ethanol
Image slices using a confocal microscope. When possible, image and quantify slices that have visible electrode marks to identify the recording plane within the slice to assess differences between electrophysiological conditions