Locating Gene Mutations Associated With Breast Cancer Using IHC (ImmunoHistoChemistry)

1. Locating Gene Mutations Associated With Breast Cancer Using IHC (ImmunoHistoChemistry) http://www.abcam.co.jp/nqo1-antibody-ab34173.html

2. Purpose • IHC can be used to detect mutations in genes that are associated with the development of breast cancer, and the risk of developing breast cancer. • IHC can also detect HER2 hormone receptors on the tumors surface, which rates it on a scale form 0 to 3+.

3. Purpose • IHC tests can tell a persons risk for developing breast cancer, pinpoint the specific mutated gene, and detect the levels of hormone receptors. • All of these results greatly impact the patients treatment and preventative care.

4. Gene Mutations Related to Increased Risk of Breast Cancer • BRCA1 • BRCA2 • CDH1 • PTEN • STK11 • TP53

5. Gene Mutations Associated With Breast Cancer • AR • ATM • BARD1 • BRIP1 • CHEK2 • DIRAS3 • ERBB2 • NBN • PALB2 • RAD51 • RAD50

6. BRCA1 in Human Breast Cancer www.rndsystems.com/images/ihc/af2210_2.jpg

7. BRCA1 • Detection of Breast Cancer gene 1 (BRCA1) in paraffin-embedded human breast cancer tissue sections using 5 µg/mL of R&D Systems goat anti-human affinity-purified BRCA1 polyclonal antibody. Tissues were stained using anti-goat HRP-DAB detection reagents (brown) and counterstained with hematoxylin (blue). • This antibody specifically recognizes human BRCA1. This antibody has not yet been tested in other species. This antibody can be used for immunohistochemistry, immunoprecipitation, Western blot, and direct ELISA.

8. Troubleshooting

9. Overstating

10. High Background

11. Procedure Sample Preparation (Frozen Tissues) The vast majority of immunohistochemical procedures employ a cell or tissue fixation step using formaldehyde or other cross-linking fixatives prior to incubation with primary antibody. Fixation is required to retain tissue morphology and prevent degradation of tissue antigens. Fixation may be performed either by immersing dissected pieces of tissue (e.g. human biopsies) into the fixative, or by vascular perfusion (e.g. laboratory animals such as mice, rats, guinea pigs, etc.). It is very important to optimize fixing conditions since under- or over-fixation may reduce or abolish tissue immunoreactivity. The easiest way to correct under-fixation is to post-fix tissue sections on the slide before starting immunohistochemical staining. To recover antigens in over-fixed tissues, either protease-induced epitope retrieval (PIER) or heat-induced epitope retrieval (HIER) techniques are recommended. HIER can be performed using a microwave oven, pressure cooker, vegetable steamer, autoclave or water bath. After tissues are fixed, they may either be embedded into paraffin or covered with OCT compound and frozen for further sectioning. Paraffin-embedded tissues are cut using a microtome at room temperature, whereas frozen tissues are cut using a cryostat at temperatures below 0° C. Antigen immunoreactivity was found to be better preserved in frozen rather than paraffin-embedded tissues.

12. Procedure Tissue Fixation and Mounting: • Fix the tissue by vascular perfusion with 500 - 700 mL of Fixative (-10% Neutral Buffered Formalin) • Dissect the tissue. • Immerse the tissue in 70% ethanol three times for 30 minutes each at room temperature. • Immerse the tissue in 90% ethanol two times for 30 minutes each at room temperature. • Immerse the tissue in 100% ethanol three times for 30 minutes each at room temperature. • Immerse the tissue in toluene three times for 20 minutes each at room temperature. • Embed the tissue in paraffin (Paraplast, Fisher Scientific) two times for 60 minutes each at 58° C. Alternatively, tissues can be embedded into paraffin using specialized automated tissue processing systems. • Cut 5 - 15 µm thick tissue sections using a rotary microtome. • Float the sections in a 56° C water bath. • Mount the sections onto histological slides. • Dry the slides overnight at room temperature. Slides containing paraffin-embedded sections can be stored at room temperature.

13. Procedure • When it is not possible to fix tissue by perfusion, dissected tissue may be fixed by immersing the tissue into a 10% formalin solution for 4 - 8 hours at room temperature. It is commonly accepted that the volume of fixative should be 50 times greater than the size of the immersed tissue. Avoid fixing the tissue for greater than 24 hours since tissue antigens may either be destroyed or masked (A.C. Cuello, ed., 1993, Immunohistochemistry: Methods in the Neurosciences, Vol. 14; IBRO Handbook Series, John Wiley & Sons, New York).

14. Procedure Antigen Retrieval Before Staining: Most formalin-fixed tissue requires an antigen retrieval step before immunohistochemical staining can proceed. This is due to the formation of methylene bridges during fixation, which cross-link proteins and therefore mask antigenic sites. The two methods of antigen retrieval are heat-mediated (also know as heat-induced epitope retrieval, or HIER) and enzymatic. Antigen retrieval with Tris/EDTA pH 9.0 buffer is suitable for most antigens. Sodium citrate pH 6.0 is also widely used.

15. Procedure • Insert the slides containing tissues into the heated Retrieval Solution and incubate 2 - 10 minutes. Note: cryostat sections are more susceptible to the damaging effects of the Retrieval Solution than paraffin-embedded tissues. • Place the Coplin jar containing the Retrieval Solution and slides on a lab bench and allow to cool for 5 - 10 minutes at room temperature. • Rinse the slides with distilled water followed by PBS. Note: tissues may become loose after the retrieval procedure, avoid vigorous rinsing to prevent detachment of the tissues from the slides.

16. Procedure Staining: Day 1 • Wash the slides 2 x 5 minutes in TBS plus 0.025% Triton X-100 with gentle agitation. • Block in 10% normal serum with 1% BSA in TBS for 2 hours at room temperature • Drain slides for a few seconds (do not rinse) and wipe around the sections with tissue paper • Apply primary antibody diluted in TBS with 1% BSA • Incubate overnight at 4°C

17. Procedure Day 2 1. Rinse 2 x 5min TBS 0.025% Triton with gentle agitation. 2. If using an HRP conjugate for detection, incubate the slides in 0.3% H2O2 in TBS for 15 min 3. For enzymatic detection (HRP-DAB): Apply enzyme-conjugated secondary antibody to the slide diluted to the concentration recommended by the manufacturer in TBS with 1% BSA, and incubate for 1 hour at room temperature. 4. Rinse 3 x 5min TBS. 5. Develop with chromogen for 10 min at room temperature 6. Rinse in running tap water for 5 min. 7. Counterstain, if desired, with hematoxylin for 1-5 seconds. Slides are then air dried and can be mounted and coverslipped with Immunohistochemistry Mounting Medium without any further dehydration. Slides that need to be kept for long periods in optimal shape can be dehydrated in ethanol and mounted in special mounting medium. 8. Dehydrate, clear and mount

18. References • http://www.jhc.org/cgi/rapidpdf/jhc.7A7209.2007v1.pdf • http://ghr.nlm.nih.gov/condition=breastcancer • http://www.breastcancer.org/symptoms/testing/types/ihc.jsp • http://www.abcam.com/ps/pdf/protocols/ihc_p.pdf • http://www.rndsystems.com/ihc_detail_objectname_EnzymaticProtocol20.aspx