Free-floating IHC/IF for thick neural tissue sections
Materials Required
Principle
Free-floating immunohistochemistry (IHC) and immunofluorescence (IF) for thick neural tissue sections involve staining tissue sections suspended in solution to enhance antibody penetration, particularly beneficial for thick sections (40-80 μm) used in 3D reconstruction and stereology. This method improves uniformity of labeling and reduces tissue loss compared to slide-mounted techniques.
MCE has not independently verified the accuracy of these methods. They are for reference only.
Experimental Materials
• Paraformaldehyde-fixed brain tissue
• Vibratome or cryostat for sectioning
• Free-floating staining trays
• Primary and secondary antibodies (species-specific)
• Blocking agents (e.g., serum or BSA)
• PBS or TBS buffer
• Detergent (e.g., Triton X-100)
• DAB or fluorophore-conjugated secondary antibodies
• Mounting media, coverslips
• Optional: antigen retrieval solutions (e.g., citrate buffer), pressurized incubation devices, and RNAscope probes for dual ISH-IHC
• Vibratome or cryostat for sectioning
• Free-floating staining trays
• Primary and secondary antibodies (species-specific)
• Blocking agents (e.g., serum or BSA)
• PBS or TBS buffer
• Detergent (e.g., Triton X-100)
• DAB or fluorophore-conjugated secondary antibodies
• Mounting media, coverslips
• Optional: antigen retrieval solutions (e.g., citrate buffer), pressurized incubation devices, and RNAscope probes for dual ISH-IHC
Experimental Procedure
• 1. Perform intra-aortic perfusion with paraformaldehyde followed by brain dissection.
• 2.Section brain tissue using a vibratome or cryostat to obtain thick sections (40-80 μm).
• 3. Incubate sections in blocking solution (e.g., 5% normal serum in PBS + 0.3% Triton X-100) for 1-2 hours at room temperature.
• 4. Incubate with primary antibody diluted in blocking solution overnight at 4°C or 37°C (optimized per protocol).
• >5. Wash sections 3× with PBS/Triton X-100.
• 6. Incubate with fluorophore-conjugated secondary antibody for 2-4 hours at room temperature.
• 7. Wash thoroughly.
• 8. Mount sections on glass slides with antifade mounting medium and cover with coverslip.
• 9. For multiplex labeling, use sequential incubation with different primary antibodies from the same host species using immunoglobulin depletion or other strategies.
• 10. For enhanced penetration in thick tissues, consider pressurized immunohistochemistry or incubation at 37°C.
11.Image using confocal or fluorescence microscopy.
• 2.Section brain tissue using a vibratome or cryostat to obtain thick sections (40-80 μm).
• 3. Incubate sections in blocking solution (e.g., 5% normal serum in PBS + 0.3% Triton X-100) for 1-2 hours at room temperature.
• 4. Incubate with primary antibody diluted in blocking solution overnight at 4°C or 37°C (optimized per protocol).
• >5. Wash sections 3× with PBS/Triton X-100.
• 6. Incubate with fluorophore-conjugated secondary antibody for 2-4 hours at room temperature.
• 7. Wash thoroughly.
• 8. Mount sections on glass slides with antifade mounting medium and cover with coverslip.
• 9. For multiplex labeling, use sequential incubation with different primary antibodies from the same host species using immunoglobulin depletion or other strategies.
• 10. For enhanced penetration in thick tissues, consider pressurized immunohistochemistry or incubation at 37°C.
11.Image using confocal or fluorescence microscopy.
Troubleshooting
Poor antibody penetration
May result from overly thick sections (>100 μm)Use thinner sections or apply pressure-assisted incubation.
High background staining
Can be reduced by increasing blocking time, optimizing antibody dilution, or including more detergent.Non-specific binding
May be mitigated by using pre-absorbed antibodies or testing antibody specificity.Fluorescence quenching
Can be prevented by using antifade mounting media and minimizing light exposure during processing.References:
- [1]. Potts EM, et al. Histological-Based Stainings using Free-Floating Tissue Sections. J Vis Exp. 2020;162:e61622.&. [Content Brief]
- [2]. Tu L, Zhang N, Conde K, et al. Free-floating Immunostaining of Mouse Brains. J Vis Exp. 2021;176:e62876.& [Content Brief]
- [3]. Kelly AM, et al. Protocol for multiplex fluorescent immunohistochemistry in free-floating rodent brain tissues. STAR Protoc. 2022;3(4):101672.&. [Content Brief]
- [4]. Xiao X, Feng YP, Du B, et al. Antibody incubation at 37°C improves fluorescent immunolabeling in free-floating thick tissue sections. Biotechniques. 2017;62(3):115-122.& [Content Brief]
- [5]. Piekut DT, et al. Penetration of immunoreagents in Vibratome-sectioned brain: a light and electron microscopic study. J Histochem Cytochem. 1983;31(5):669-674.&. [Content Brief]
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