Whole mount cryosections were prepared for immunofluorescence microscopy as previously described (Ridky et al., 2010 (link)). Briefly, slides were fixed in 4% paraformaldehyde or −20°C methanol, permeabilized as required and blocked with 5% goat serum/PBS, followed by incubation with primary antibodies, and secondary antibodies conjugated to fluorophores. Slides were mounted with Prolong Gold Antifade Reagent with DAPI (Life Technologies, Grand Island, NY). The primary antibodies used in this study were IQGAP1 and Collagen VII (Millipore, Billerica, MA), IQGAP3 (Abcam, Cambridge, MA), Desmogelin-3 (Invitrogen, Carlsbad, CA), and Keratin-5 (Covance, Conshohocken, PA).
Protocol detail
Immunofluorescence Microscopy of Cryosections
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Antibodies
Collagen
Cryosections
Dapi
Goat
Gold
Immunofluorescence microscopy
Keratin 5
Methanol
Paraformaldehyde
Serum
Corresponding Organization :
Other organizations : University of Pennsylvania
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Variable analysis
independent variables
- Fixation method (4% paraformaldehyde or -20°C methanol)
- Immunofluorescence staining of IQGAP1, IQGAP3, Collagen VII, Desmogelin-3, and Keratin-5
- Permeabilization as required
- Blocking with 5% goat serum/PBS
- Incubation with primary and secondary antibodies
- Mounting with Prolong Gold Antifade Reagent with DAPI
- No explicit positive or negative controls were mentioned.
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