PRODUCT-SPECIFIC PROTOCOLS
| IHC (HSPD1) | |
|---|---|
| Sample type: | human intestinal cancer tissue, human lung cancer tissue, human mammary cancer tissue, mouse liver tissue, rat liver 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: | 0.5-1μg/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.
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