Jan 31, 2025

Public workspaceFluorescence immunohistochemistry for quantitative imaging and analysis

DOI
dx.doi.org/10.17504/protocols.io.81wgbrq4qlpk/v1
  • Andrew D. Sauerbeck1,
  • Terrance T. Kummer1
  • 1Department of Neurology, Hope Center for Neurological Disorders, Knight Alzheimer's Disease Research Center, Washington University School of Medicine, St. Louis, MO 63110, USA
Maria Matos
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DOI: dx.doi.org/10.17504/protocols.io.81wgbrq4qlpk/v1
Protocol Citation: Andrew D. Sauerbeck, Terrance T. Kummer 2025. Fluorescence immunohistochemistry for quantitative imaging and analysis. protocols.io https://dx.doi.org/10.17504/protocols.io.81wgbrq4qlpk/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 31, 2024
Last Modified: January 31, 2025
Protocol Integer ID: 118333
Keywords: ASAPCRN
Funders Acknowledgements:
Aligning Science Across Parkinson's
Grant ID: ASAP-020616
Abstract
Quantitative imaging and analysis of fluorescently stained tissue sections is sensitive to multiple factors. Two critical ones are endogenous autofluorescence and careful preparation of antibodies. This protocol provides methods for reducing the impact of autofluorescence through high-intensity broad wavelength light exposure. Additionally, it outlines a stepwise protocol for creating primary antibody Master Mixes which are then subsequently combined together, or with buffer, to ensure consistency in antibody concentrations across all conditions.
Materials
Reagents:

ABC
ReagentVendorCat#
10x Stock PBSCorningMT-46013CM
Normal Goat SerumVectorS-1000-20
GlycineSigma AldrichG7126-1KG
AcetamideSigma AldrichA0500-500G
Sodium AzideSigma AldrichS2002-100G
Ethylene GlycolSigma Aldrich102466-1L
SucroseFisher ChemicalS5-500
10000K Finisher BulbHTG SupplyLAM-AM24T5AK-B
Mowiol 4-88Electon Microscopy Sciences17977-150
AF-300Electon Microscopy Sciences17977-25
1.5H coverglassMarienfeld107242
DAPIInvitrogenD3571

Reagent 10X Phosphate-Buffered SalineCorningCatalog #46-013-CM
ReagentNormal Goat SerumVector LaboratoriesCatalog #S-1000-20
ReagentGlycineMerck MilliporeSigma (Sigma-Aldrich)Catalog #G7126
ReagentAcetamideMerck MilliporeSigma (Sigma-Aldrich)Catalog #A0500-500G
ReagentSodium AzideMerck MilliporeSigma (Sigma-Aldrich)Catalog #S2002-100G
ReagentAgroMax 2 Foot T5 Finisher Bulb – 10000KHTG SupplyCatalog #LAM-AM24T5AK-B
ReagentCitifluor Mwl4-88Electron Microscopy SciencesCatalog #17977-150
ReagentCitifluor Af300Electron Microscopy SciencesCatalog #17977-25
ReagentDAPIInvitrogen - Thermo FisherCatalog #D3571

Light Treatment and Blocking
Light Treatment and Blocking
2d 1h 10m
2d 1h 10m
Remove formaldehyde fixed brain cryosections sections from storage plates containing cryopreservation solution (30% Sucrose + 30% Ethylene Glycol + 0.02% Sodium Azide in 1X PBS).
Rinse tissue sections three times in 1X Phosphate Buffered Saline (PBS) for Duration00:05:00 each.

5m
Transfer sections to 48 well tissue culture plate containing 1x PBS.

Carefully remove all PBS and replace with ‘Light Treatment’ buffer (1x PBS + 4% Glycine + 4% acetamide + 0.05% Sodium Azide).

Ensure all sections are flat on the bottom of each well.

Seal wells containing tissue sections with clear Duck Brand EZ-Start tape.

Turn on high-intensity light bulbs (AgroMax T5 Finisher Bulb – 10000k) and ensure that the intensity of light at the surface of each bulb is greater than 400 Lux. Re-measure Lux every Duration24:00:00 .

Position the tissue plate such that each section is positioned directly above the bulb.

Expose the tissue sections to >400 Lux light for Duration40:00:00 -Duration48:00:00 at TemperatureRoom temperature .

2d
Rinse tissue sections three times in 1X PBS for Duration00:05:00 each.

Block tissue sections for Duration01:00:00 at TemperatureRoom temperature with 20% NGS + 0.3% TritonX-100 + 0.02% Sodium Azide in 1X PBS.

  • Blocking buffer must be centrifuged at Centrifigation17500 x g, 00:05:00 before use.

1h 5m
Centrifigation
Primary Antibody Preparation
Primary Antibody Preparation
3d 0h 10m
3d 0h 10m
Make ‘Master mix’ of each primary antibody such that the antibody concentration is 3x greater than the final target working concentration. Combine Master mixes with each other or primary antibody buffer to create antibody cocktails for Experimental tissue sections and No Primary controls. Primary antibody buffer is 10% normal goat serum (NGS) + 0.3% TritonX-100 + 0.02% Sodium Azide in 1X PBS .

Thoroughly mix all solutions using brief pulses on a bench top vortex.

Centrifuge all final antibody mixes at Centrifigation17500 x g, 00:05:00 .

5m
Centrifigation
Remove all 1X PBS from wells with tissue sections and replace with primary antibody mixture.

Seal wells with tissue sections with clear Duck Brand EZ-Start tape.

Place tissue sections on shaker at TemperatureRoom temperature , protect from light, and incubate primary antibodies for Duration64:00:00 -Duration72:00:00 .

3d
Incubation
Rinse tissue sections three times in 1X PBS for Duration00:05:00 each.
5m
Secondary Antibody and DAPI Staining
Secondary Antibody and DAPI Staining
1d 0h 55m
1d 0h 55m
Create mixture of secondary antibodies in 10% NGS + 0.3% TritonX-100 + 0.02% Sodium Azide in 1X PBS.

Centrifuge all final secondary mixes at Centrifigation17500 x g, 00:05:00 .

5m
Centrifigation
Remove all 1X PBS from wells with tissue sections and replace with secondary antibody mixture.

Seal wells with tissue sections with clear Duck Brand EZ-Start tape.

Place tissue sections on shaker at TemperatureRoom temperature , protect from light, and incubate secondary antibodies for Duration18:00:00 -Duration24:00:00 .

1d
Incubation
Rinse tissue sections three times in 1X PBS + 0.3% TritonX-100 for Duration00:15:00 each.

15m
Remove PBS + TritonX-100 and replace with DAPI in 1X PBS at a concentration of 1:50,000.

Incubate tissue sections in DAPI for Duration00:10:00 -Duration00:20:00 .

20m
Incubation
Rinse tissue sections two times in 1X PBS for Duration00:15:00 each.

15m
Mounting
Mounting
Mount tissue sections on charged histology slides in 1X PBS.

Remove excess PBS from around tissue sections and then allow slides to dry at a 45-degree angle protected from light.

Immediately when sections have fully dried, quickly dip the sections 5 times in deionized water to rinse off excess salts.

Remove excess water from around tissue sections and then allow slides to dry at a 45-degree angle protected from light.

Immediately when sections have fully dried, apply Mowiol 4-88 mounting media that has been freshly prepared the day of staining. The Mowiol mounting is made by mixing Mowiol 4-88 with AF-300 in a 9:1 ratio. The mixture must be thoroughly vortexed and then centrifuged to remove all bubbles. Apply Amount300 µL -Amount600 µL of mountant mixture to the slide.

Apply thoroughly cleaned 1.5H coverglass with gentle pressure to expel excess Mowiol from underneath the coverglass.

Allow the slides to dry horizontally protected from light for at least Duration24:00:00 . The mountant will continue to harden over time and 3-7 days of curing is recommended to ensure solid curing and improved refractive index matching.

Protocol references
Sauerbeck AD, Gangolli M, Reitz SJ, Salyards MH, Kim SH, Hemingway C, Gratuze M, Makkapati T, Kerschensteiner M, Holtzman DM, Brody DL, Kummer TT. SEQUIN Multiscale Imaging of Mammalian Central Synapses Reveals Loss of Synaptic Connectivity Resulting from Diffuse Traumatic Brain Injury. Neuron. 2020 Jul 22;107(2):257-273.e5. doi: 10.1016/j.neuron.2020.04.012. Epub 2020 May 8. PMID: 32392471; PMCID: PMC7381374.