PRODUCT-SPECIFIC PROTOCOLS

ICC/IF (CAST)
Sample type:	U20S cell
Cell preparation:	4% Paraformaldehyde
Permeabilization:	0.1% Triton X-100 for 10 minutes at RT.
Blocking buffer:	10% goat serum
Primary antibody concentration:	2μg/ml
Incubation time:	overnight at 4°C
Detection Method:	fluorescence microscope

PROTOCOL

Cell Climbing Slice Preparation

Use tweezers to take out coverslip from well plate, wash with PBS for three times to remove culture medium.
Immerse coverslip (cells face up) into 4% Paraformaldehyde or neutral formalin for 10-20 min (close the lid to prevent volatilization). Wash the coverslip with PBS for three times.
Put coverslips on filter paper (cells face up). Remove the liquid on the coverslip and allow it to dry for 8-10 hours.

Permeabilization

Immerse the cells in 0.1% Triton X-100 for 10 minutes at room temperature.

Blocking

Add 10% normal goat serum reagent.
Incubate the samples at 37°C for 30 min.
Discard extra liquid (no washing required).

Primary Antibody Incubation

Dilute primary antibody with antibody diluent to the concentration recommended by the antibody manufacturer.
Add the diluted antibody to the samples and incubate overnight at 4°C or at 37°C for 1 hour.
Wash the samples 3X with PBS.

Secondary Antibody Incubation

Fluorophore-conjugated Goat Anti-Rabbit IgG at the appropriate ratio to the samples and incubate at 37°C for 30 min.
Wash the samples 3X with PBS.

Staining

The section was counterstained with DAPI.
Visualize using a fluorescence microscope and filter sets appropriate for the label used.