Jan 31, 2025
Fluorescence immunohistochemistry for quantitative imaging and analysis
- 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
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, fluorescence immunohistochemistry for quantitative imaging, fluorescence immunohistochemistry, fluorescence, endogenous autofluorescence, autofluorescence, impact of autofluorescence, creating primary antibody master mix, careful preparation of antibody, primary antibody master mix, antibody concentration, antibody, consistency in antibody concentration, immunohistochemistry, quantitative imaging, analysis quantitative imaging, light exposure, stained tissue section, intensity broad wavelength light exposure, imaging
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:
| A | B | C | |
| Reagent | Vendor | Cat# | |
| 10x Stock PBS | Corning | MT-46013CM | |
| Normal Goat Serum | Vector | S-1000-20 | |
| Glycine | Sigma Aldrich | G7126-1KG | |
| Acetamide | Sigma Aldrich | A0500-500G | |
| Sodium Azide | Sigma Aldrich | S2002-100G | |
| Ethylene Glycol | Sigma Aldrich | 102466-1L | |
| Sucrose | Fisher Chemical | S5-500 | |
| 10000K Finisher Bulb | HTG Supply | LAM-AM24T5AK-B | |
| Mowiol 4-88 | Electon Microscopy Sciences | 17977-150 | |
| AF-300 | Electon Microscopy Sciences | 17977-25 | |
| 1.5H coverglass | Marienfeld | 107242 | |
| DAPI | Invitrogen | D3571 |
10X Phosphate-Buffered SalineCorningCatalog #46-013-CM
Normal Goat SerumVector LaboratoriesCatalog #S-1000-20
GlycineMerck MilliporeSigma (Sigma-Aldrich)Catalog #G7126
AcetamideMerck MilliporeSigma (Sigma-Aldrich)Catalog #A0500-500G
Sodium AzideMerck MilliporeSigma (Sigma-Aldrich)Catalog #S2002-100G
AgroMax 2 Foot T5 Finisher Bulb – 10000KHTG SupplyCatalog #LAM-AM24T5AK-B
Citifluor Mwl4-88Electron Microscopy SciencesCatalog #17977-150
Citifluor Af300Electron Microscopy SciencesCatalog #17977-25
DAPIInvitrogen - Thermo FisherCatalog #D3571
Troubleshooting
Light Treatment and Blocking
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 00:05:00 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 24:00:00 .
Position the tissue plate such that each section is positioned directly above the bulb.
Expose the tissue sections to >400 Lux light for 40:00:00 -48:00:00 at Room temperature .
2d
Rinse tissue sections three times in 1X PBS for 00:05:00 each.
Block tissue sections for 01:00:00 at Room temperature with 20% NGS + 0.3% TritonX-100 + 0.02% Sodium Azide in 1X PBS.
- Blocking buffer must be centrifuged at 17500 x g, 00:05:00 before use.
1h 5m
Primary Antibody Preparation
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 17500 x g, 00:05:00 .
5m
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 Room temperature , protect from light, and incubate primary antibodies for 64:00:00 -72:00:00 .
3d
Rinse tissue sections three times in 1X PBS for 00:05:00 each.
5m
Secondary Antibody and DAPI Staining
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 17500 x g, 00:05:00 .
5m
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 Room temperature , protect from light, and incubate secondary antibodies for 18:00:00 -24:00:00 .
1d
Rinse tissue sections three times in 1X PBS + 0.3% TritonX-100 for 00:15:00 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 00:10:00 -00:20:00 .
20m
Rinse tissue sections two times in 1X PBS for 00:15:00 each.
15m
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 300 µL -600 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 24:00:00 . 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.