Apr 29, 2025
IHC from Frozen Section
This protocol is a draft, published without a DOI.
- 1University of Maryland School of Medicine
Protocol Citation: Prajwal Ciryam 2025. IHC from Frozen Section. protocols.io https://protocols.io/view/ihc-from-frozen-section-drv6569e
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: November 18, 2024
Last Modified: April 29, 2025
Protocol Integer ID: 112286
Keywords: IHC, Frozen Section
Funders Acknowledgements:
GEn1E Lifesciences
Abstract
The purpose of this protocol is to outline the steps in performing immunohistochemistry (IHC) from frozen sections of tissue, primarily mouse and rat. The mounted microscope slides generated from this protocol can be directly processed via imaging on a fluorescent or confocal microscope.
Materials
1xPBS
ImmunoPen
Slide chamber
Kimwipes
2% donkey serum
0.2% Triton-X 100
Aluminum foil
dH2O
Prolong Gold Anti-Fade Mountant with DAPI
Microscope slides
Microscope slide coverslips
Safety warnings
N/A
Ethics statement
This protocol needs prior approval by the users' Institutional Animal Care and Use Committee (IACUC) or equivalent ethics committee.
Prepare Sections
Prepare Sections
55m
55m
Take slides of brain sections out from -20 °C storage and bring slides to room temperatures.
10m
Make sure slides are dry, devoid of moisture and solution droplets.
Wash the slides in PBS for 5 minutes x 2 to wash off OCT in a slide chamber on a nutator.
10m
Wipe off the PBS around the brain sections using small Kimwipes but do not touch the sections themselves.
Use an ImmunoPen to make wells around the sections. Make sure the pen marking dries before proceeding.
5m
Wash in PBS for 10 minute x 3 in a slide chamber on a nutator.
30m
Dry the slides using Kimwipes.
Blocking
Blocking
1h
1h
Block for 1 hour at room temperature. This should be done in a humidified slide tray.
- Blocking solution is typically 2% donkey serum, 0.2% Triton-X in PBS.
- Serum should be based on the species of your secondary antibody.
1h
Gently tap the remaining solution off the slides on a dry paper towel.
Primary Antibody
Primary Antibody
30m
30m
Prepare primary antibody at the appropriate dilution in blocking solution. General primary antibody dilutions range from 1:100 to 1:500, depending on the antibody.
Incubate overnight at 4ºC.This should be done in a humidified slide tray.
Alternatively, incubate 1h at room temperature, or 48h at 4ºC. Different incubation methods can be combined.
Wash slides in PBS x 3 for 10 minutes each in a slide chamber at room temperature on a nutator.
30m
Wipe off major droplets around the section using Kimwipes. Do not let the tissue dry as this will cause background fluorescence.
Secondary Antibody
Secondary Antibody
2h 1m
2h 1m
Prepare secondary antibody at 1:500 in blocking solution. From this step forward avoid the light.
Incubate for 1 hour at room temperature. This should be done in a humidified slide tray
1h
Wash the slides in PBS x 3 for 10 minutes each in a slide chamber at room temperature, covering the slide chamber with aluminum foil to protect samples from light.
30m
Quickly rinse the slides by dipping them in a clean slide chamber filled with distilled or deionized H2O.
1m
Wipe off excess water from around the tissue.
Allow the tissue to dry completely under aluminum foil.
30m
Mount
Mount
Add Prolong Anti-Fade Mountant with DAPI using a 1000 µL pipette tip with the end of the tip cut off. Avoid bubbles.
Place rectangular coverslip onto slide by placing it perpendicular to the slide near the bottom edge and then slowly lowering it onto the slide.
Inspect the slide for bubbles and if they are overlying the tissue, then push gently on the coverslip with tweezers/forceps to force the bubbles away. Be careful not to damage the tissue. Sometimes it is best to leave good enough alone.
Slides can be stored at room temperature, protected from light.
Protocol references
Protocol originally adapted from Sveta Ivanova.