Home › Immunology › Detection of Modified Forms of Cytosine Using Sensitive Immunohistochemistry
Immunology JoVE (Open Access) Citable · DOI

Detection of Modified Forms of Cytosine Using Sensitive Immunohistochemistry

DOI: 10.3791/54416-v
What you'll learn
  • ✓ Prepare tissue sections for immunostaining
  • ✓ Perform immunostaining for oxidized cytosine derivatives
  • ✓ Apply tyramide signal amplification for sensitivity
  • ✓ Analyze spatial distribution in brain tissues
Protocol

Biopharma Insights Herein we describe a sensitive immunochemical method for mapping the spatial distribution of 5mC oxidation derivatives based on the use of peroxidase-conjugated secondary antibodies and tyramide signal amplification.

Difficulty
advanced
Total time
~1 day
Model organism
Mouse
Biosafety
BSL-1

Steps

1
Fix and permeabilize tissue sections

Tissue sections from cryo- or microtome are fixed and permeabilized to prepare for antibody staining.

▶ 00:47
2
Immunostain for oxi-5mC derivatives

Apply primary antibodies specific to oxidized forms of 5mC, followed by peroxidase-conjugated secondary antibodies and tyramide signal amplification.

▶ 01:34
3
Analyze 5hmC and 5caC distributions

Visualize the immunostaining results to characterize the distribution of 5hmC and 5caC in adult mouse brain tissues.

▶ 05:16
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