Thymi were harvested and embedded in Tissue-Tek Optimal Cutting Temperature media. Eight micrometer frozen thymic sections were fixed in 100% acetone and blocked in 10% goat serum before incubation with primary antibodies. Primary antibodies were purchased from either Abcam (keratin-5, keratin-8) or eBioscience (Aire) and all secondary antibodies were purchased from Invitrogen. Immunofluorescence slides were visualized using a Zeiss Apotome widefield microscope. For eye disease scoring, eyes were processed by formalin fixation and H&E staining as previously described [6 (link), 8 (link)]. Sections were blindly scored for severity of infiltration and tissue destruction. H&E slides were imaged using a Zeiss AxioImager brightfield microscope.
Protocol detail
Immunofluorescence and Histology of Thymus and Eyes
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Acetone
Aire
Antibodies
Eye disease
Eyes
Formalin
Frozen sections
Goat
Immunofluorescence
Keratin 5
Keratin 8
Microscope
Serum
Thymi
Thymic
Tissue
Corresponding Organization : University of California, San Francisco
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Variable analysis
independent variables
- Thymic tissue harvesting
- Embedding in Tissue-Tek Optimal Cutting Temperature media
- Acetone fixation of frozen thymic sections
- Blocking in 10% goat serum
- Incubation with primary antibodies (keratin-5, keratin-8, Aire)
- Immunofluorescence visualization using Zeiss Apotome widefield microscope
- Eye disease severity scoring (infiltration and tissue destruction) using H&E stained sections and Zeiss AxioImager brightfield microscope
- Thymic section thickness (8 micrometers)
- Primary antibodies purchased from Abcam and eBioscience
- Secondary antibodies purchased from Invitrogen
- Not explicitly mentioned
- Not explicitly mentioned
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