Adenomatous Polyposis Coli (APC) Immunohistochemistry Kit For Immunohistochemical Staining of Adenomatous Polyposis Coli (APC) in human FFPE Tissue RUK-KAP01-20 For Research Use Only Riverside Biosciences Inc. 2327 S 5th Ave, North Riverside, IL60546 Tel: (312)685-11598; Email:info@rbiosciences.com
Introduction Adenomatous Polyposis Coli (APC) is a tumor suppressor protein that acts as an antagonist of the Wnt signaling pathway. It is also involved in other processes including cell migration and adhesion, transcriptional activation, and apoptosis. Defects in this gene cause familial adenomatous polyposis (FAP), an autosomal dominant premalignant disease that usually progresses to malignancy. Disease-associated mutations tend to be clustered in a small region designated the mutation cluster region (MCR) and result in a truncated protein product. Diseases associated with APC also include cranial nerve palsy, and desmoid tumor. APC promotes rapid degradation of CTNNB1 and participates in Wnt signaling as a negative regulator. Kit Contents Reagents provided in the kit The materials listed are sufficient for 20 tests. The number of tests is based on the use of 200 μl each of ready to use reagent per slide. Positive Control Slides (Cat# KC-PC-RCCX001) One human renal carcinoma slides Blocking Buffer (Cat# KC-BB-001) 400μl 10X Non-specific blocking buffer Dilute at 1:10 using distilled water prior to staining; unused working solution may be stored at 4 C for 3 month. Equilibrium Buffer (Cat# KC-EB-001) 4mL Equilibrium Buffer Ready to use reagent Mouse anti-human Adenomatous Polyposis Coli (APC) antibody (Cat# KC-APC-001) 80μl Mouse anti-adenomatous Polyposis Coli (APC) antibody Dilute in Antibody Diluents immediately before use (recommend use at 1:50 dilution). Antibody Diluent (Cat# KC-AD-001) 4mL Antibody Dilutent Ready to use reagent Wash Buffer (20x, Cat# KC-WB-001) 16mL, Tris buffered saline with Tween 20 (ph7.6) Dilute at 1:20 using distilled water prior to staining; unused working solution may be stored at 4 C for 3 month. Mouse HRP Polymer (Cat# KC-MH-001) 4mL Mouse HRP Polymer Ready to use reagent DAB substrate buffer (Cat# KC-DS-001) 0.4 ml 10X DAB substrate buffer
Hydrogen Peroxide (H2O2) for DAB substrate buffer (Cat# KC-HP-001) 60uL, 0.3% Hydrogen Peroxide solution DAB Chromogen (Cat# KC-DC-001) 100uL, Diaminobenzedinetetrahydrochloride (DAB) substrate solution (Do not expose DAB components to direct or bright light during storage and staining process). Materials required but not included in the kit Reagents: Xylene Ethanol Endogenous Peroxidase Blocking Solution (3% Hydrogen Peroxide) Hematoxylin Mounting media Distilled or deionized water Antigen Retrieval Buffer (10X) 0.1M Citrate Buffer (ph6.0) Lab Equipment: Steamer or microwave oven (for antigen retrieval) PAP pen for restraining reagents on slides Moist chamber for slides incubation with staining reagents General lab equipment for immunohistostaining such as slide racks, staining jars, cover slips, timer, pipettes, etc. Microscope equipment and accessories Storage and stability Store Adenomatous Polyposis Coli (APC) IHC Kit at 2 8 C. The kit is stable for six months at 4 C. Do not use after expiration date. Precautions Take reasonable precautions when handling reagents. Use disposable gloves when handling suspected carcinogens or toxic materials (examples: DAB, xylene and H2O2). Unused solution should be disposed according to applicable local, state and federal regulations. Protocol The Adenomatous Polyposis Coli (APC) Immunohistostaining Kit has been designed for the staining of tissues that have been fixed (usually in neutral buffered formalin) and subsequently embedded in paraffin before sectioning. This protocol is recommended as a starting point and optimization by the individual end user may be required. Note: Do not allow specimens to dry during the staining procedure. Specimen drying may cause increased non specific staining and background.
Some tissue may need to bake to remove over covered paraffin prior to the procedure. If needed, bake at 55 60 C for 30 minutes. I. Deparaffinization and rehydration Prior to staining, tissue sections must be deparaffinized and rehydrated. Incomplete removal of paraffin can cause poor staining of the section. Use positive control slide provided in the kit for quality control and trouble-shooting purpose. 1. Immerse slides in xylene and incubate for 5 minutes. Repeat twice with fresh xylene for another 5 minutes each. 2. Immerse slides in 100% ethanol for 5 minutes, and follow with immersion in 95%, 75% and 50% ethanol for 3 minutes each. 3. Rinse slides with distilled water for 5 minutes; keep in water until ready to perform antigen retrieval. II. Heat induced antigen retrieval (HIAR) Most formalin fixed tissue requires an antigen retrieval step before immunohistochemical staining can proceed. Heat induced antigen retrieval can be performed using a steamer, pressure cooker, or a microwave oven. The retrieval time written in this protocol is based on using a steamer. The heating time may need to be adjusted if you use a different device and method. 1. Fill plastic coplin jar/container with Antigen Retrieval Buffer (0.01M Citrate Buffer, ph6.0, not included in the kits). Prepare Stock Solution: 0.1M Sodium Citrate 20.5mL 0.1M Citric Acid 4.5mL Add distilled water to 250mL 2. Place the coplin jar/container in steamer with lid. 3. Turn on steamer and preheat to 90 100 C. Carefully put slides into the coplin jar/container and steam for 40 min (95 100 C). 4. Turn off the steamer, remove the coplin jar, place at room temperature and allow slides to cool for 20 min. Keep the jar covered all the time. 5. Rinse slide by incubation of slide in distilled water for 3 minutes. Repeat this step twice and begin staining procedure. III. Staining procedure Blocking of Endogenous Peroxidase Note: Peroxidase Blocking is optional. If no non-specific staining is observed, skip these steps and go to step 3. 1. Tap off excess water. Draw a circle around the specimen on the slide with PAP pen (not included in the kit. Alternatively, folded Kimwipes could be used to briefly blot the water around the specimen. Repeat this blot step each time before add reagent on slide). Apply 200μl or more of Peroxidase
Blocking Solution (not included in the kit) sufficient to cover specimen, and incubate for 5 minutes. 2. Rinse slide by incubation of slide in distilled water for 3 minutes. Repeat this step twice. 3. Rinse slide by incubation slide in PBS for 3 minutes. Blocking of Non-specific binding 4. Tap off excess PBS. (If the Peroxidase Blocking step is skipped, draw a circle around the specimen on the slide with PAP pen or using the edge of folded Kimwipes to quickly blot the water around the specimen). Apply 200μl 1X Blocking Buffer immediately to cover specimen and incubate in a moist chamber for no more than 10 minutes Note: 10X blocking buffer may form precipitates at 4 C. Completely dissolve the precipitates before making working solution 5. Rinse slide by incubation slide in PBS for 3 minutes. Primary Antibody 6. Tap off excess PBS. Apply 200μl Equilibrium Buffer immediately to cover specimen and incubate in a moist chamber for 30 minutes 7. Tap off excess Equilibrium Buffer. Apply 200μl diluted anti Adenomatous Polyposis Coli (APC) antibody (recommend 1:50 dilution in Antibody Diluent) to cover specimen immediately and incubate in a moist chamber overnight at 4 C. 8. Rinse slide by incubation in 0.5-2mL Wash Buffer for 3 minutes. Repeat this step twice with fresh buffer. 9. Rinse slide by incubation of slide in PBS for 3 minutes. Secondary/HRP Conjugates 10. Tap off excess PBS. Apply 200μl Mouse HRP Polymer immediately to cover specimen and incubate in a moist chamber for 60 minutes. 11. Rinse slide by incubation in 0.5-2mL Wash Buffer for 3 minutes. Repeat this step twice with fresh buffer. 12. Rinse slide by incubation in PBS for 3 minutes. DAB Chromogen 13. Tap off excess PBS. Apply enough DAB Substrate Solution to cover specimen immediatly. Check dark brown color development under microscope and incubate until desired stain intensity develops. To make 1mL DAB Substrate Solution, mix the following reagents: Distilled Water 860 μl 10X DAB substrate buffer 100 μl 0.3% Hydrogen Peroxide solution 15 μl DAB Chromogen 25 μl 14. Rinse slide in tap water for 3 minutes.
Counterstaining 15. If desired, complete counterstain (See instruction for hematoxylin counterstaining). Rinse in tap water to clear. Mounting 16. Immerse slides in 70%, 80%, 95% Ethanol for 2 minutes each, and 100% Ethanol for 10 minutes twice followed by Xylene for 5 minutes twice. 17. Dry and mount slides. IV. Instruction for Hematoxylin counterstaining 1. Immerse slides in hematoxylin solution. Incubate for 30 seconds to 5 minutes, depending on the strength of hematoxylin used. 2. Rinse to clear with tap water and continue dehydration from Step 16. Image shows immunohistochemical staining of paraffin-embedded mouse pancreatic section with APC antibody using the Riverside Biosciences APC IHC Kit (Cat No. KAP01-20). APC (dark brown) displays a plasma membrane/cytosol localization pattern (20X, counterstained with hematoxylin).
Troubleshooting Problems Possible Causes Solutions Over staining Weak or no staining High background Too long incubation time of primary antibody, or too high temperature when doing staining Too long incubation time of DAB substrate. Slide dried during staining process Incomplete removal of paraffin Tissues over fixation Not efficient antigen retrieval Reagents not used in proper order or omitted steps Expired antibody or reagents Sections dried during staining process Slide not rinsed thoroughly Antigen over retrieval Depending on tissue sections, the incubation time of primary antibody can be reduced to 2 hours; Check the room temperature range is at 20 25 C when doing staining. Reduce incubation time of DAB substrate Avoid sections to dry during staining process. Deparaffinize sections longer or change to fresh xylene; some tissue array may need to bake to remove over covered paraffin. Increasing the concentration of primary antibody to 1:40; if this does not work, reduce duration of post fixation. Adjust antigen retrieval time based on the setting for section fixation and retrieval device used. Review notes and procedure used. Check kit expiration dates and kit storage conditions Do not allow sections to dry during staining process; use humid container during incubation with primary antibody. Use fresh solution in buffer jars; rinse at least three times between steps. Optimize antigen retrieval time if you used microwave or pressure cooker for retrieval.