Free floating brain sections were stained for CYP3A4, GFAP, NeuN, and MDR1. Adjacent sections were stained with CV (Marchi et al., 2004 (link), 2006 (link); Moddel et al., 2005 (link)). Two antibodies against CYP3A4 were used: (1) rabbit polyclonal anti-human CYP3A4 (AB1254) (1:1,000; Millipore); (2) rabbit polyclonal anti-human CYP3A4 (CR 3340, 1:1,000, BIOMOL; Enzo Life sciences, U.S.A.). We also used: mouse monoclonal anti-GFAP (G 3893, 1:100; Sigma, MO, U.S.A.); mouse monoclonal anti-NeuN (1:500; Millipore) and mouse monoclonal anti-MDR1 (1:40, C494; Calbiochem, U.S.A.). Secondary antibodies were chosen according to the primary host: Texas red dye–conjugated Affinipure donkey anti-mouse IgG (1:100; Jackson Immunoresearch Laboratories Inc.) and fluorescein isothiocyanate (FITC)-conjugated Affinipure donkey anti-rabbit IgG (1:100; Jackson Immunoresearch Laboratories Inc.). Auto fluorescence was blocked with Sudan Black B. Sections were mounted using Vectashield mounting medium with DAPI (Vector).
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Immunohistochemical Staining of Brain Sections
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Anti igg
Antibodies
Brain
Cyp3a4
Dapi
Donkey
Fluorescein
Fluorescence
Gfap
Human
Isothiocyanate
Mouse
Rabbit
Sudan black b
Vector
Corresponding Organization :
Other organizations : Institute of Molecular Medicine, Cleveland Clinic, Neurological Surgery
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Variable analysis
independent variables
- Staining of brain sections with different antibodies: CYP3A4, GFAP, NeuN, MDR1
- Fluorescence intensity and localization of CYP3A4, GFAP, NeuN, and MDR1 in brain sections
- Adjacent brain sections stained with Cresyl Violet (CV)
- Two different antibodies used for CYP3A4 staining
- Secondary antibodies used: Texas red for mouse IgG, FITC for rabbit IgG
- Auto-fluorescence blocked with Sudan Black B
- Sections mounted with DAPI-containing medium
- Positive control: Adjacent brain sections stained with Cresyl Violet (CV)
- Negative control: Not explicitly mentioned
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