PRODUCT-SPECIFIC PROTOCOLS

IF (VEGFA)
Sample type:	human breast cancer tissue, human endometrial cancer tissue, human ovarian cancer tissue, human pancreatic cancer tissue, human prostatic cancer tissue, human thyroid cancer tissue
Sample preparation:	4% Paraformaldehyde
Antigen retrieval:	citrate buffer (pH6.0) or EDTA buffer (pH 8.0)
Blocking buffer:	10% goat serum
Incubation time:	overnight at 4°C
Detection Method:	Chromogenic Reagents

PROTOCOL

Tissue Preparation

Paraformaldehyde cooling and dehydration.
Liquid paraffin section.
Section slicing and incubation.

Deparaffinization

Immerse the slides in xylene 2 times for 10 minutes each.
100% ethanol (for 10 min) - 95% ethanol (for 10 min) - 70% ethanol (for 10 min) - 50% ethanol (for 10 min).
Wash the slides with water to remove ethanol.

Antigen Retrieval

Immerse the paraffin sections in EDTA buffer.
Heat the buffer in microwave for 5-8 min.
Cool the slides until it reaches room temperature.
Wash the sections 1X to 2X with PBS.

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 the primary antibody with antibody diluent according to the dilution ratio recommended in the table.
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.
Add the secondary antibody diluted 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.