Visualizing Extracellular and Membrane Proteins
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Corresponding Organization : Université Paris-Sud
Other organizations : University of California, Irvine, New York University, Cohen Children's Medical Center, Northwell Health, Sorbonne Université, Centre d'Immunologie et des Maladies Infectieuses, Centre National de la Recherche Scientifique
Variable analysis
- Tissue preparation method (flash freezing in OCT at -80°C, cryosectioning into 12- to 14-μm-thick sections)
- Localization and visualization of extracellular and membrane proteins
- Localization and visualization of internal antigens
- Nuclear localization of β-Cat
- Sirius Red and fluorescence intensities (ΔCTCF) in threshold-matched images
- Paraformaldehyde fixation of sections
- Blocking with 1% bovine serum albumin (BSA), 1:10 anti-mouse CD16/CD32 Fc block
- Incubation with appropriate antibodies in 1% BSA/PBS/Fc block for 2 hours at 4°C
- Incubation with appropriate antibodies in PBS containing fish gelatin and saponin overnight
- Permeabilization with 0.3% Triton X-100 for nuclear localization of β-Cat
- Mounting with Vectashield DAPI (4′,6-diamidino-2-phenylindole)
- Positive control: Not explicitly mentioned
- Negative control: Not explicitly mentioned
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