3ColorStaining:AlphaCatenininHeLaHumanCervicalCancerCells

實驗概要

Alpha-catenin  in HeLa human cervical cancer cells was labeled using mouse  anti-α-catenin and visualized with Alexa Fluor? 488 goat anti-mouse IgG  (green). Filamentous actin was visualized using red-fluorescent Alexa  Fluor? 635 phalloidin. Nuclear DNA was stained with blue-fluorescent  DAPI.

主要試劑

10X Phosphate-buffered saline (PBS) [詳細信息：GIBCO?]

Triton X-100 [詳細信息：Sigma]

Methanol-free 16% formaldehyde solution [詳細信息：Polysciences]

Bovine serum albumin [詳細信息：Sigma]

Normal goat serum (10%) [詳細信息：Invitrogen Cat. no. 50-19Z]

Anti-α-catenin antibody, mouse monoclonal [詳細信息：Invitrogen Cat. no 180225]

Alexa Fluor? 488 goat anti-mouse IgG

Alexa Fluor? 635 phalloidin

DAPI

Prolong? Gold antifade reagent

實驗材料

Confluent HeLa human cervical cancer cells

實驗步驟

(All steps are carried out at room temperature unless otherwise noted.)

1. Sample Preparation

1) Remove media from live cells and fix cells for 15 minutes in 4% formaldehyde solution.

2) Wash cells 3 x 5 minutes in PBS (1X).

3) Permeabilize cells by treating for 10 minutes with PBT (0.1% Triton X-100 in PBS).

4) Wash cells 3 x 5 minutes in PBS.

2. Cell Staining

1) Block cells for 1 hour in antibody blocking buffer (10% normal goat serum (NGS), 1% BSA in PBT).

2) Incubate cells with 5 μg/ml of anti--catenin primary antibody in antibody blocking buffer for 45 minutes.

3) Wash cells 3 x 10 minutes in PBT.

4) Incubate cells with 5 g/ml Alexa Fluor? 488 goat anti-mouse IgG in 1% BSA/PBT for 30 minutes.

5) Wash 3 x 5 minutes in PBS.

6) Incubate cells for 20 minutes with Alexa Fluor? 635 phalloidin, diluted 1:40 from stock solution.

7) Wash 3 x 5 minutes in PBS.

8) Mount the specimen in ProLong? Gold antifade reagent.

3. Mounting Reagent Preparation and Sample Processing

1) Remove the ProLong Gold antifade reagent from the freezer and allow the vial to equilibrate to room temperature.  NOTE:   Using an external heat source to warm the vial is not recommended, as this may decrease the long-term stability of the product.

2) Remove  any excess liquid from the specimen and apply 1 or 2 drops (depending  on the surface area of your sample) of the antifade reagent to the  specimen.

3) Cover  slide-mounted specimens with a coverslip; for specimens mounted on  coverslips, place a drop of antifade reagent onto a clean slide and  carefully lower the coverslip onto the antifade reagent to avoid  trapping any air bubbles.

4) Allow  the mounted sample to cure on a flat surface in the dark. Curing time  may vary from a couple of hours to overnight, depending on the thickness  of the sample and the relative humidity of the surrounding air.

5) For  long-term storage, seal the coverslip to the slide after curing to  prevent excessive shrinkage of the mounting medium, which can result in  sample distortion. After sealing, store the slide upright in a covered  slide box at <–20°C. Desiccant may be added to the box to ensure that  the slide remains dry.

6) To  view the samples immediately, secure the coverslip at the corners using  nail polish or hot wax to prevent the coverslip from moving. Leave the  edges clear to allow the preparation to cure.

注意事項

1)         The antifade properties of ProLong? Gold antifade reagent improve slightly the longer it remains in contact with the specimen.

2)         To  further reduce photobleaching, minimize the exposure of fluorescently  labeled specimens to light by using neutral density filters, and expose  samples only when observing or recording a signal. Optimize image  capture by using a minimum of optics, high-numerical aperture  objectives, relatively low magnification, high-quality optical filters,  and high-speed film or high-efficiency detectors.

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