Locating Gene Mutations Associated With Breast Cancer

Using IHC (ImmunoHistoChemistry)

http://www.abcam.co.jp/nqo1-antibody-ab34173.html

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+.

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.

Gene Mutations Related to Increased Risk of Breast Cancer

• BRCA1• BRCA2• CDH1• PTEN• STK11• TP53

Gene Mutations Associated With Breast Cancer

• AR• ATM• BARD1• BRIP1• CHEK2• DIRAS3• ERBB2• NBN• PALB2 • RAD51• RAD50

BRCA1 in Human Breast Cancer

www.rndsystems.com/images/ihc/af2210_2.jpg

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.

TroubleshootingPossible Source Test or Action

Lack of antigen Check protein expression by in situ hybridization (in some rare cases translation may be blocked even

though mRNA is detected)

Antibodies do not work due to improper storage

Aliquot antibodies into smaller volumes sufficient to make a working solution for a single experiment. Store aliquots in a freezer (-20 to -70°C) and avoid repeated

freeze-thaw cycles

Inactive primary antibodies Replace with a new batch of antibodies

Inadequate tissue fixation Try different fixative

Incompatible secondary and primary antibodies

Use secondary antibody that will interact with the primary antibody. For example, if primary antibodies

were raised in rabbits, use anti-rabbit secondary but not anti-mouse or anti-goat

Inactive secondary reagents Use secondary antibody that will interact with the primary antibody. For example, if primary antibodies

were raised in rabbits, use anti-rabbit secondary but not anti-mouse or anti-goat

Inactive secondary reagents Replace with new reagents

Antigen was destroyed before incubation with primary antibody

Quenching of endogenous peroxidase was done prior to the addition of primary antibodies. Block peroxidase

after incubation with primary antibody

Overstating Possible Source Test or Action

High concentration of primary and/or secondary antibodies and/or reagent

Determine optimal concentration for each component of immunohistochemical reaction: primary antibodies; secondary antibodies; enzymes catalyzing formation of color precipitates

Long incubation time Determine optimal incubation time for each component of immunohistochemical reaction: primary antibodies; secondary antibodies; enzymes; chromogenic substrates

Non-specific binding of primary and/or secondary reagents to tissues

Treat tissues to reduce or block non-specific binding of immunohistochemical components to tissues, for example, by Avidin-Biotin Blocking Reagents

High Background

Possible Source Test or Action

Tissues have endogenous molecules which are also used in incubation mixtures, for example, the presence of peroxidase in blood cells or the presence of autofluorescent pigment lipofuscin in neuronal cells

Incubate with normal serum obtained from species other than from the species that were the source of the primary antibody

Block endogenous component. Endogenous peroxidase may be blocked by incubating tissues for 30 minutes at room temperature with 0.3% H2O2 in methanol, or switch to protocols utilizing substances which are not present in tissues

For autofluorescence: after finishing IHC staining, treat tissues with 1% Sudan Black in 70% alcohol. If residual autofluorescence still obscures specific labeling, use non-fluorescent enzymatic protocols with chromogenic substrates (for instance DAB or AEC) or employ immunogold-silver staining

ProcedureSample 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.

ProcedureTissue Fixation and Mounting:1. Fix the tissue by vascular perfusion with 500 - 700 mL of Fixative (-10% Neutral

Buffered Formalin)2. Dissect the tissue. 3. Immerse the tissue in 70% ethanol three times for 30 minutes each at room

temperature. 4. Immerse the tissue in 90% ethanol two times for 30 minutes each at room

temperature. 5. Immerse the tissue in 100% ethanol three times for 30 minutes each at room

temperature. 6. Immerse the tissue in toluene three times for 20 minutes each at room temperature. 7. 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.

8. Cut 5 - 15 µm thick tissue sections using a rotary microtome. 9. Float the sections in a 56° C water bath. 10. Mount the sections onto histological slides. 11. Dry the slides overnight at room temperature. Slides containing paraffin-embedded

sections can be stored at room temperature.

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).

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 pH6.0 is also widely used.

Procedure

1. 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.

2. 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.

3. 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.

Procedure

Staining:Day 1 1. Wash the slides 2 x 5 minutes in TBS plus 0.025% Triton X-100

with gentle agitation. 2. Block in 10% normal serum with 1% BSA in TBS for 2 hours at

room temperature 3. Drain slides for a few seconds (do not rinse) and wipe around the

sections with tissue paper 4. Apply primary antibody diluted in TBS with 1% BSA 5. Incubate overnight at 4°C

ProcedureDay 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 bythe 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

References • http://www.jhc.org/cgi/rapidpdf/jhc.7A7209.2

007v1.pdf• http://ghr.nlm.nih.gov/

condition=breastcancer• http://www.breastcancer.org/symptoms/testi

ng/types/ihc.jsp

• http://www.abcam.com/ps/pdf/protocols/ihc_p.pdf

• http://www.rndsystems.com/ihc_detail_objectname_EnzymaticProtocol20.aspx