Mar 29, 2026
Immunohistochemistry Protocol for Brain Sections
- Sayan Dutta1
- 1California Institute of Technology
Protocol Citation: Sayan Dutta 2026. Immunohistochemistry Protocol for Brain Sections. protocols.io https://dx.doi.org/10.17504/protocols.io.eq2ly47rplx9/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: August 12, 2025
Last Modified: March 29, 2026
Protocol Integer ID: 224523
Keywords: immunohistochemistry protocol for brain sections immunohistochemistry, brain sections immunohistochemistry, subcellular expression patterns in situ, brain section, immunohistochemistry protocol, specific proteins within tissue, subcellular expression pattern, combining antibody, fluorescent detection, specific protein
Abstract
Immunohistochemistry on brain sections is used to visualize and localize specific proteins within tissue, combining antibody labeling with fluorescent detection. This method enables analysis of cellular and subcellular expression patterns in situ.
Materials
Phosphate-buffered saline (PBS), pH 7.4
Triton X-100 (Sigma-Aldrich #T8787 or equivalent)
Normal donkey serum (Jackson ImmunoResearch #017-000-121)
Primary antibodies (as per experimental design; host species as appropriate)
Fluorophore-conjugated secondary antibodies (Alexa Fluor series, 1:500; Jackson ImmunoResearch, West Grove, PA)
ProLong‱ Diamond Antifade Mountant (Thermo Fisher Scientific #P36970)
Microscope slides (Brain Research Laboratories, Cat# 2575-PLUS, Superfrost Plus)
Glass coverslips (standard No. 1.5 thickness; Fisher Scientific or equivalent)
Parafilm for covering sections during incubation
Slide storage box for air-drying overnight
Forceps and fine brushes for handling sections
Light-protected containers for incubations with fluorescent antibodies
Troubleshooting
Begin with free-floating brain sections stored in PBS containing 0.02% sodium azide to preserve tissue integrity. Gently transfer the sections into fresh PBS supplemented with 1% Triton X-100 and incubate for 1 hour at room temperature. This step permeabilizes the tissue, allowing antibodies to access intracellular targets.
After permeabilization, place the sections into a blocking solution consisting of 10% normal donkey serum prepared in PBS with 0.3% Triton X-100 (PBST). Incubate the tissue in this blocking buffer for 90 minutes at room temperature to reduce nonspecific antibody binding and improve staining specificity.
Prepare the primary antibody solution in PBST containing 1% normal donkey serum. Transfer the sections into this solution and incubate them overnight at 4 °C with gentle agitation. This extended incubation ensures thorough antibody penetration and binding to the target epitopes.
On the following day, carefully wash the sections three times in PBS, each wash lasting 10 minutes, to remove any unbound primary antibody.
Incubate the washed sections with fluorophore-conjugated secondary antibodies (Alexa Fluor series, diluted 1:500 in PBST with 1% normal donkey serum) for 90 minutes at room temperature, protected from light. This step allows visualization of the primary antibody targets.
Once secondary labeling is complete, wash the sections again three times in PBS for 10 minutes each to remove excess secondary antibody and reduce background fluorescence.
Mount the stained sections onto clean glass microscope slides and allow them to air-dry overnight in the dark. Once dry, apply coverslips using ProLong™ Diamond Antifade Mountant to preserve fluorescence and prevent photobleaching during imaging.