Thymi were harvested and embedded in O.C.T. media (Tissue-Tek). 8-µm frozen thymic sections were fixed in 100% acetone, blocked, and stained for keratin-5, keratin-8 (Abcam), or Aire (eBioscience). Secondary antibodies were purchased from Invitrogen. Thymic sections were visualized using a widefield microscope (Apotome; Carl Zeiss). For tumor immunostaining, tumors were harvested and fixed in 10% formalin as previously described (Zhu et al., 2013 (link)). Fixed tumors were embedded in paraffin and sectioned for staining with anti-CD3 antibody and counterstained with DAPI. Tumor sections were visualized using a fluorescent microscope (BX60; Olympus) and analyzed using ImageJ software (National Institutes of Health).
Protocol detail
Analyzing Thymic and Tumor Sections
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Acetone
Aire
Anti antibody
Antibodies
Dapi
Embedded paraffin
Formalin
Frozen sections
Keratin 5
Keratin 8
Microscope
Thymi
Thymic
Tissue
Tumor
Corresponding Organization : University of California, San Francisco
Other organizations : University of North Carolina at Chapel Hill
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Variable analysis
independent variables
- Harvesting and embedding thymi in O.C.T. media (Tissue-Tek)
- Fixing thymic sections in 100% acetone
- Blocking thymic sections
- Staining thymic sections with antibodies for keratin-5, keratin-8, and Aire
- Harvesting and fixing tumors in 10% formalin
- Embedding and sectioning fixed tumors
- Staining tumor sections with anti-CD3 antibody
- Visualization of thymic sections using a widefield microscope (Apotome; Carl Zeiss)
- Visualization of tumor sections using a fluorescent microscope (BX60; Olympus)
- Analysis of tumor sections using ImageJ software (National Institutes of Health)
- Thickness of thymic sections (8-µm)
- Fixation of tumors in 10% formalin as previously described (Zhu et al., 2013)
- Positive controls: Not specified
- Negative controls: Not specified
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