PRODUCT-SPECIFIC PROTOCOLS

IHC (CCAR2)
Sample type:	human gall bladder adenosquamous carcinoma tissue, human liver cancer tissue, human lung cancer tissue, mouse brain tissue, rat brain tissue, human laryngeal carcinoma tissue, human pancreatic carcinoma tissue, human renal clear cell carcinoma tissue, rat brain tissue
Tissue preparation:	Paraffin-embedded Section
Antigen retrieval:	citrate buffer (pH6.0) or EDTA buffer (pH 8.0)
Blocking buffer:	10% goat serum
Primary antibody concentration:	1-2 μg/ml
Incubation time:	overnight at 4°C
Detection Method:	DAB

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.

Inactivation

Immerse dewaxed paraffin section into the 3% H₂O₂ at room temperature for 10 min.
Wash the section 3X to 5X with distilled water (total 3 to 5 min).

Antigen Retrieval

Immerse the paraffin sections in EDTA buffer or citrate buffer (pH 6.0).
Heat the buffer in microwave for 5-8 min.
Cool the slides until it reaches room temperature.

Blocking

Add 10% normal goat serum to the HIER treated samples.
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.
Wash the samples 3X with PBS (5 min each).

Secondary Antibody Incubation

Dilute the HRP-conjugated secondary antibody 1:500 with antibody diluent.
Add the diluted antibody to the samples and incubate at 37°C for 30 min.
Wash the samples 3X with PBS (5 min each).

Staining

Add a suitable amount of DAB reagent to the samples and incubate in dark at room temperature for 5 to 30 min.
Monitor the tissue staining intensity under a bright-field microscope.
Wash the samples 3X to 5X with distilled water.
Counterstain (if necessary).
Dehydrate, permeabilization (optional) and mount.