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Protocol
Immunofluorescence stain of cells in Permanox chambers
Keywords: Permanox, Chamber slides, adherent, cell culture, hank´s
Permanox is a specific type of polymer classified as a member of the polyolefin family. Permanox Chamber Slide Products are cell culture treated for adherent cell culture.
Required Materials
- PBS , with 1% w/v BSA
- Triton X
- PBS , with 0,1% Triton-X
- BSA
- Primary antibody , Golgi, mouse monoclonal anti-58k (1:50; ab6284, AbcamUK)
- Primary antibody , ER, rabbit polyclonal anti-calreticulin (1:50; NB600-101 Novus Biologicals)
- Lab-Tek ™ Slides , with 8 chambers and lid.
- Primary antibody , Mitoch, mouse monoclonal [II-14-10] anti-prohibitin (ab1836, abcam)
- Primary antibody , Vesicles, mouse monoclonal [X22] anti-clathrin (1:250 Ab2731, membrane vesicle marker, Abcam)
- Secondary antibody , Alexa Fluor® 546 goat anti-mouse IgG1 ( 1) *2 mg/mL (1:800; Molecular Probes Inc.)
- Secondary antibody , AlexaFlour 488 goat anti-rabbit IgG highly cross-adsorbed (1:2,000; Molecular Probes Inc., Eugene)
- Shandon PermaFluor™
- Mounting media
- Milli-Q water
- DAPI
- PBS , or Hank´s buffer
- Cells , from culture to be analyzed.
- Cover slips
- Lab-Tek ™ Slides , with 4 chambers and lid.
- Zeiss Axiovision , for inspection of slides.
Main steps
- Preparation of Hank´s buffer
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Mount with water soluble Fluorescence Mounting Medium from DakoCytomation.…
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Seed colon HCT116 / SW480 cells on 8 well Permanox chamber slides (Nunc)…
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Wash cells three times with warm PBS or with Hank´s buffer.
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Fixate cells by gentle immersion in acetone, at −20°C for 10 min. …
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Wash cells three times in PBS.
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Permeabilize the fixed cells with 0.1% Triton X-100/PBS for 30 min.
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Block background with PBS containing 1% w/v BSA for 45 minutes at room…
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Wash cells three times in PBS.
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Incubate with primary antibodies for 1 h at room temperature.
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Wash cells three times with PBS.
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Incubate with Alexa-Fluor 488 conjugated anti-rabbit secondary antibody.…
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Wash cells three times with PBS.
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Nuclear counter stain with DAPI or Hoechst and wash twice with PBS.
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Remove chambers and silicone rubber gasket with a scalpel and wash the…
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Mount with water soluble Fluorescence Mounting Medium from DakoCytomation.…
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Inspect the slides on a Zeiss Axiovision.
Instructions
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Preparation of Hank´s buffer
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8 well chambers on slide are 0,8cm2 / well For colon SW480 cells 4000-8000-16000 cells are recommended, depending on the experiment to be performed.
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Seed colon HCT116 / SW480 cells on 8 well Permanox chamber slides (Nunc) for 24 h to 48 h to 70-80% confluence.
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Wash cells three times with warm PBS or with Hank´s buffer.
-
Fixate cells by gentle immersion in acetone, at −20°C for 10 min. Alternately, in methanol, at −20°C for 5 min.
-
Wash cells three times in PBS.
-
Permeabilize the fixed cells with 0.1% Triton X-100/PBS for 30 min.
-
Block background with PBS containing 1% w/v BSA for 45 minutes at room temperature.
-
Wash cells three times in PBS.
-
Incubate with primary antibodies for 1 h at room temperature.
-
Wash cells three times with PBS.
-
Incubate with Alexa-Fluor 488 conjugated anti-rabbit secondary antibody.
A negative control should be done with secondary antibody only.
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Wash cells three times with PBS.
-
Nuclear counter stain with DAPI or Hoechst and wash twice with PBS.
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Remove chambers and silicone rubber gasket with a scalpel and wash the slides 3 x with Milli-Q-H2O (removal of salt and fluorescence).
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Mount with water soluble Fluorescence Mounting Medium from DakoCytomation.
Permanox® slides will warp if coverslipped using a mounting medium that contains toluene or xylenol. PERMAFLUOR™ (Lipshaw, Inc.) is recommended.
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Inspect the slides on a Zeiss Axiovision.
