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Protocol: Immunohistochemical staining (IHC)

MATERIAL

Tissue sections

Primary antibody

Conjugated secondary antibody

ABC reagents

Xylene

100% ethanol

95% ethanol

3% hydrogen peroxide

B U F FER 5

PBS

Antigen retrieval buffer: 10 mM sodium citrate buffer pH 6.0

1 mM EDTA pH 8.0

Pepsin

Blocking buffer: 5-10% normal animal serum

METHOD1:IHC-P (for paraffin embedded tissue)

I. Oeparaffinize/hydrate sections

1. Xylene: twice, each for 5 min

2. 100% ethanol: twice, each for 3 min

3. 95% ethanol : 3 min

4. 70 % ethanol: 3 min

5. 50 % ethanol : 3 min

6. Wash slides in deionized water for 3 min

II. Antigen Unmasking

1. Bring slides to a boil in antigen retrieval solution for 15 min in a microwave. Cool slides on bench top for 20 min, or

2. Pepsin digest for 10 min at 3TC.

Your Expertise, Our Antibodies, Accelerated Discovery . USA Tel: 1.949.553.1900 Toll-free: 1.877.436.3839 International Tel: 886.3.6208988

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Protocol: Immunohistochemical staining (lHC)

III. Staining

1. Wash sections in dH20 twice, each for 3 min.

2. Incubate sections in 3% hydrogen peroxide for 10 min.

3. Wash section in PBS twice, each for 3 min.

4. Block each section with blocking buffer for 20 min at RT.

5. Remove blocking buffer and add primary antibody at proper dilution and incubate for one hour at RT or over­night at 4 "C.

6. Remove antibody solution and wash sections in PBS three times, each for 3 min.

7. Add secondary antibody, and incubate for 30 min at RT.

8. (Prepare ABC reagent according to the manufacturer's instructions and mix solution for 30 min at RT).

9. Remove secondary antibody solution and wash sections in PBS three times, each for 3 min.

10. Add ABC reagent to each section and incubate for 30 min at RT.

11. Remove ABC reagent and wash sections in PBS three times ,each for 3 min.

12. Add DAB to each section and monitor staining closely.

13. As soon as the sections develop, immerse slides in dH20.

14. Counter-stain sections in hematoxylin.

15. Wash sections in dH20 twice, each for 3 min.

16. Dehydrate sections:

Incubate sections in 95% ethanol for twice, each for 3 min.

Incubate sections in 100% ethanol twic,e each for 3 min.

Incubate sections in xylene twice, each for 3 min.

17. Mount sections with coverslip.

Method 2: IHC-F (for frozen tissue)

1. Fix section by immersing in acetone jar for 1-2 min at RT, let air dry.

2. Wash sections in dH20 for twice, each for 3 min.

3. Incubate sections in 0.3% hydrogen peroxide for 10 min.

4. Wash section in PBS for twice each for 3 min.

5. Block each section with blocking solution for 20 min at RT.

6. Remove blocking solution and add primary antibody at proper dilution and incubate for one hour at RT or

Your Expertise, Our Antibodies, Accelerated Discovery . USA Tel: 1.949.553.1900 Toll-free: 1.877.436.3839 International Tel: 886.3 .6208988

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Protocol: Immunohistochemical staining (lHC)

overnight at 4'C.

7. Remove antibody solution and wash sections in PBS three times, each for 3 min.

8. Add secondary antibody, and incubate for 30 min at RT.

9. (Prepare ABC reagent according to the manufacturer's instructions and mix solution for 30 min at RT).

10. Remove secondary antibody solution and wash sections in PBS three times, each for 3 min.

11. Add ABC reagent to each section and incubate for 30 min at RT.

12. Remove ABC reagent and wash sections in PBS three times, each for 3 min.

13. Add DAB to each section and monitor staining closely.

14. As soon as the sections develop, immerse slides in dH20.

15. Counter-stain sections in hematoxylin

16. Wash sections in dH20 twice, each for 3 min.

17. Dehyd rate sections:

Incubate sections in 95% ethanol twice, each for 3 min.

Incubate sections in 100% ethanol twice, each for 3 min.

Incubate sections in xylene twice, each for 3 min.

18. Mount sections with coverslip.

Your Expertise, Our Antibodies, Accelerated Discovery . USA Tel: 1.949.553.1900 Toll-free: 1.877.436.3839 International Tel: 886.3.6208988

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GeneTex Starting as a s all scientist-founded company in San Antonio,

Texas, GeneTex flas grown to become one of the top anUJody

manufacturers and as solidified its position in the life ciences

industry.

Our corporate mission, to provide our customers with quality reagents and to accelerate life sciences research,

reflects the philosophy and approach we employ when we manufacture our products. Through extensive

research, development, and quality-assurance testing, we have produced and validated a comprehensive

collection of antibodies and research reagents .

Due to GeneTex's experience in antibody

production, we are uniquely capable of

providing technical support and

troubleshooting assistance for

our customers .

Our Antibodies

Your Expertise, Our Antibodies, Accelerated Discovery.

Antigen Analysis

Protein Expression . in E coli System / Peptide

. Synthesis

Protein Purification / Peptide

coniugatiO*

USA Tel: 1.949.553.1900 Toll-free: 1.877.436.3839 International Tel: 886.3.6208988

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Selection you need

Comprehensive Coverage

GeneTex currently offers more than 55,000 products including over 45,000 primary antibodies that cover

eleven different research areas. We continue to build upon our product portfolio to ensure that novel targets

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In addition to our comprehensive catalog of primary antibodies, GeneTex also offers a wide variety of quality

reagents, kits and services.

%

•••••••••• Angiogenesis

••••••••• Apoptosis

••••••••• Blood related

•••••••••••• Cancer related

••••••••• CDmarker

•••••••• Cell communication

••••••• _ Cell cycle

••••••• Disease related

•••••••••• DNA replication/repair/recombination

•••••••• Gene expression I ____ GPCR

•••••••••• Growth factor

••••••••••• Housekeeping gene

Immune system

•••••• Ion transport

•••••••••• Kinase and phosphatase

••••••••• Membrane transport

••••••••• Metabolism

•••••••••• Neuroscience

•••••••• Protein folding and degradation

••••••••• Signal transduction

•••••••••• Stemcell

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Your Expertise, Our Antibodies, Accelerated Discovery . USA Tel: 1.949.553.1900 Toll-free: 1.877.436.3839 International Tel: 886.3.6208988