日本細胞生物学会Japan Society for Cell Biology

Immunohistochemistry of paraffin sections

AuthorAkihiro Harada
AffiliationDepartment of Cell Biology, Graduate School of Medicine, Osaka University
Contactinquiry about this protocol
Home Pagehttp://www.med.osaka-u.ac.jp/pub/acb/eng.html
Keyword
Published2015-08-26Last Update2015-08-26
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Summary

IHC of paraffin sections (protocol from Taketo Lab, Kyoto university)

Methods

Detail  *Click photo to see a large size.

    • deparaffin
      DDW 2min×2
    • 0.01M citrate buffer 150ml Micro wave 2 min→wait 5min→microwave 30sec→wait 5min →microwave 30sec (that is, microwave 3 times)
    • leave it until the temperature goes down to the RT 1hr
    • 3%H2O2/MeOH RT 5min
      PBS  5min×2
      10%NGS 3%BSA/PBS 37℃ 1hr(30min〜3hr)
    • primary antibody in 10%NGS 3%BSA/PBS 4℃ O/N or RT 1hr
    • PBS 5min×2
      secondary antibody (1/200) in 10%NGS 3%BSA/PBS Goat anti rabbit IgG RT30min
      PBS 5min×2
    • ABC solution RT 30min(A 5μℓ B 5μℓ PBS 240μℓ )
    • PBS 5min×2
    • DAB+substrate 30μℓ/㎖ RT for a few minutes to ten minutes depending on the staining
    • wash with water
    • counterstain with hematoxylin a few seconds
    • wash in the water 5min〜10min
      dehydrate
      xylene
    • enclose the sample in EUKITT (O.Kindler Co., Germany)

Little trick

Reference