1. Paraffin section slides can be stored at room temperature for a long time.

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1 Immunohistochemistry (IHC) Protocols Immunohistochemistry (IHC) Protocol of Paraffin Section 1. Fix dissected tissues with 10% formalin for no less than 48 hours at room temperature. Inadequately fixation can make tissues dehydrated during tissue processing and become hard and brittle. 2. Rinse the tissue with running tap water for 30min-40min to eliminate the formaldehyde 3. Dehydrate the tissues in EtOH baths in the following order: 70% Ethanol 20 min (x1); 95% Ethanol 20 min (x2); 100% Ethanol 20 min (x2) 4. Clear the tissue in xylene for 2 times, 20 min each. 5. Melt the paraffin prior to adding the tissue. Incubate the tissue in a 65 C paraffin bath for 2 times, 30 min each. 6. Pour melted paraffin into paraffin block mold. Place the tissue well in the mold and wait for its cooling down. (15-20 min) 7. Section the paraffin-embedded tissue block in 4-10 μm thickness slides on a microtome and float in a 37 C water bath containing deionized water. 8. Float the sections onto clean glass slides and microwave at 65 C for 15 min, then the tissue binds to the glass. Slides can be stored overnight at room temperature or be used in immunohistochemical staining immediately. Advantages: 1. Paraffin section slides can be stored at room temperature for a long time. 2.Paraffin section is more suitble to reveal distribution of target antigen compared to frozen section. Disadvantages: Epitopes of target antigens are likely to be damaged by high temperature or fixative in the whole process and a antigen retrieval procedure is needed. Immunohistochemistry (IHC) Protocol of Frozen Section 1. Place the tissue in a small sealed box (if the tissue is too small, to hold it, pour in some embedding reagent like OCT); 2. Froze the box in liquid nitrogen (approximately 10-20s needed); 3. Transfer the tissue to frozen section machine or store at -80 C for future use; 4. Equilibrate at -20 C for approximately 15 minutes to prevent cracking of the tissue block when sectioning; 5. Section the block at a range of 6-8 µm and place on slides; 6. To help tissue adhere, air dry sections for a few minutes before fixing.

2 Advantages: 1. Frozen section nornally takes less time than paraffin section due to simpler procedures. 2. In frozen section process, the activity and epitope of target antigens can be well preserved compared to paraffin section, thus a antigen retrieval procedure is not usually needed in frozen section. Disadvantages: 1. The tissue could be less morphologically interacted than paraffin section and antigen signals might be more dispersed in the final scope. 2. The section slides might only be stored at low temperature like -80 C. Immunohistochemistry (IHC) Protocol of Antigen Retrieval (necessary for paraffin section) Protease-induced antigen retrieval (PIER) Antigen retrieval buffer for protease-induced antigen retrieval (epitope retrieval): 2 mg/ml pepsin in Tris-HCl ph 2.0 Or 0.5 mg/ml trypsin in Tris-HCl ph 8.0 Protease-induced antigen retrieval protocol: 1. Pre-warm the buffer to 37 C; 2. Keep the sections in retrieval buffer at 37 C for minutes. Suitable for antigens that are sheltered by fixative such as: Collagen, GFAP, Complement, Cytokeratin, C-erB-2, LCA, LN, etc. Heat-induced antigen retrieval (HIER) Antigen retrieval buffer for heat-induced antigen retrieval (epitope retrieval): 10 mm Sodium Citrate ph 6.0 Heat-induced antigen retrieval protocol: 1. Microwave the antigen retrieval buffer until boiling and power it off, put in the sections; 2. Repeat step 1 for one or two more times for every 5-10 minutes;

3 3. Cool down at room temperature and wash with dh 2 O. Preferred antigens: Bcl-2, Bax, c-myc, E-cadherin, ChromograninA, Heatshock protein, HPV, P-glycoprotein, PKC, PCNA, etc. Immunohistochemistry (IHC) Protocol of Immuno-staining 1. Deparaffinize: Wash slides with specific reagents in the following order: xylene, two times, 5 min each. 100% ethanol, two times, 5min each 95% ethanol, two times, 5 min each 80% ethanol, once, 5 min 70% ethanol, once, 5 min 50% ethanol, once, 5min dh 2 O, two times, 5 min each 2. (Recommended) Block the endogenous peroxidase activity at room temperature by a 5~10 min incubation in the final developmental 3% H 2 O 2 in distilled water or PBS (ph 7.4). 3. Rinse sections with PBS for 5 min. 4. Antigen retrieval. 5. Rinse sections with PBS for 5 min. 6. Apply the blocking antibody (normal goat serum), incubate for 20 min at room temperature, and throw off residual fluid (don't wash.). 7. Apply the primary antibody 60 min at RT or 4 C for overnight 8. Rinse sections twice for 5 min each. 9. Incubate array slide with a biotin-conjugated secondary antibody at 20~37 C for 20 min.

4 10. Rinse sections twice for 5min each. 11. Incubate slides with SABC reagent at 37 C for 20 min. 12. Rinse sections 4 times for 5 min each. 13. Proceed with chromogen of final developmental DAB or use DAB Kit (Control the degree of staining with regular microscopy). 14. Wash sections in distilled water. 15. Stain and differentiate slides in hematoxylin. 16. Dehydration and transparency of slides. 17. Mount slides. Immunohistochemistry (IHC) Protocol of Counterstaining DAPI Counterstaining: 1. Wash and equilibrate the sections with PBS for 5 min*3. 2. Dilute the DAPI solution to an appropriate concentration of working solution as the manual instruction. 3. Drop appropriate amount of DAPI working solution to the slides as the manual instruction (Normally µl) and make sure the tissue is all covered by the solution. 4. Incubate at room temperature for 5 min. 5. Wash in PBS bath for 5 min*3. 6. Mount the sections with mounting medium and coverslip. To make this step simple, mounting medium (DAPI-contained) is recommended. Haematoxylin Eosin (H&E) Counterstaining: 1. Counterstain in eosin-phloxine solution for 30 seconds to 1 minute. 2. Dehydrate through 95% alcohol, 2 changes of absolute alcohol, 5 minutes each. 3. Clear in 2 changes of xylene, 5 minutes each. 4. Mount with xylene based mounting medium. Immunohistochemistry (IHC) Protocol of Apoptosis TUNEL staining protocol for paraffin section In Situ Cell Death (Apoptosis) Detection by TUNEL labeling

5 by Boehringer Mannheim (Catalog No ), modified by Josiah N. Wilcox, José C. Rodriguez, and Héctor de Léon - Emory University - April 1996 Protocol for Paraffin Sections: 1. Dewax paraffin sections: Incubate slides, 55 C, 30 min. Xylenes, 2 times, 2 min. each 100% EtOH, 2 times, 2 min. each 95% EtOH, 2 times, 2 min. each 80% EtOH, 2 min. 75% EtOH, 2 min. 50% EtOH, 2 min. 2. dh2o rinse. 3. Incubate slides in a 1µg/ml Proteinase K/10mM Tris solution, 15 min., RT. (7.5µl of 20µg/µl PK in 150 ml 10mM Tris, ph ). 4. All slides: 1x PBS rinse, 2 times (+ 10 min for those non-positive control slides). 5. (Positive control slide: in DNase I solution (100µl of 200µg/ml), 10 min., RT. 1x PBS rinse, 2 times in a separate container then combine with other slides.) 6. Wipe around tissue. 7. Make up negative Control solution (just Label solution containing FITC) and TUNEL solutions at time of use: A. Remove 100µl from Tube 2 (Label solution) for 2 negative controls (50µl each). Do this even if you are omitting this negative control so that volumes and concentrations will remain consistent for the labeling. B. Add Total volume (50µl,) of Tube 1 (TdT) + remainder of Tube 2 (450µl). 8. Apply 100µl TUNEL reaction mixture (or 100µl Control Label solution for negative control) to each slide. 9. Incubate in humid chamber, 60 min., 37 C x PBS wash, 3 times. 11. Wipe around tissue. 12. Apply 100µl anti-fitc-ap conj. ("converter-ap") on each sample. 13. Incubate in humid chamber, 30 min., 37 C x PBS wash, 3 times mM Tris buffer, ph 8.2, 5 min., RT. 16. Add µl substrate solution (5-6 drops Vector Blue or Vector Red substrate/per slide): Mix:

6 5ml 100mM Tris, ph drop Levamisole 2 drops each of Solution 1, 2, and 3 of either Vector substrate 17. Incubate in absence of light, RT. Vector Blue - 10 min.; Vector Red min. 18. dh2o, 1 time to stop color reaction. Counterstain for Vector Blue: Gill's Hematoxylin, No. 2, 5 sec. Scott's solution, 20 sec. 70% EtOH, 30 sec. 95% EtOH, 2 times, 30 sec. each 100% EtOH, 2 times, 30 sec. each Histoclear, 1 min. Histoclear, 1 min. Coverslip with Accumount medium. Counterstain for Vector Red: Gill's Hematoxylin, No. 2, 5 sec. Scott's solution, 20 sec. 70% EtOH, 30 sec. 95% EtOH, 2 times, 30 sec. each 100% EtOH, 2 times, 30 sec. each Xylenes, 1 min. Xylenes, 1 min. Coverslip with Accumount medium. Reagents Needed: 3L 1x PBS 10mM Tris-HCl 20 mg/ml Proteinase K 100µl/slide x-phosphate/bcip or Fast Red substrate DNase I solution (1mg/ml - 1µg/ml) for Positive control 100mM Tris-Hcl, ph 8.2

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